欢迎您, 登录 | 注册

首页 | English

X
加载中

Ribosome footprinting, or Ribo-seq, has revolutionized the studies of translation. It was originally developed for yeast and mammalian cells in culture (Ingolia et al., 2009). Herein, we describe a plant-optimized hands-on ribosome footprinting protocol derived from previously published procedures of polysome isolation (Ingolia et al., 2009; Mustroph et al., 2009) and ribosome footprinting (Ingolia et al., 2009; Ingolia et al., 2013). With this protocol, we have been able to successfully isolate and analyze high-quality ribosomal footprints from different stages of in vitro grown Arabidopsis thaliana plants (dark-grown seedlings [Merchante et al., 2015] and 13-day-old plantlets in plates and plants grown in liquid culture [unpublished results]).

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

A Ribosome Footprinting Protocol for Plants
植物核糖体的足迹分析实验

植物科学 > 植物细胞生物学 > 细胞器分离
作者: Catharina Merchante
Catharina MerchanteAffiliation 1: Instituto de Hortofruticultura Subtropical y Mediterranea (IHSM)-UMA-CSIC, Departamento de Biología Molecular y Bioquímica, Universidad de Málaga, Spain
Affiliation 2: Department of Plant and Microbial Biology, North Carolina State University, Raleigh, USA
Affiliation 3: Genetics Graduate Program, North Carolina State University, Raleigh, USA
Bio-protocol author page: a3650
Qiwen Hu
Qiwen HuAffiliation: Department of Computer Science, North Carolina State University, Raleigh, USA
Bio-protocol author page: a3651
Steffen Heber
Steffen HeberAffiliation: Department of Computer Science, North Carolina State University, Raleigh, USA
Bio-protocol author page: a3652
Jose Alonso
Jose AlonsoAffiliation 1: Department of Plant and Microbial Biology, North Carolina State University, Raleigh, USA
Affiliation 2: Genetics Graduate Program, North Carolina State University, Raleigh, USA
Bio-protocol author page: a3653
 and Anna N. Stepanova
Anna N. StepanovaAffiliation 1: Department of Plant and Microbial Biology, North Carolina State University, Raleigh, USA
Affiliation 2: Genetics Graduate Program, North Carolina State University, Raleigh, USA
For correspondence: atstepan@ncsu.edu
Bio-protocol author page: a3654
Vol 6, Iss 21, 11/5/2016, 1025 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1985

[Abstract] Ribosome footprinting, or Ribo-seq, has revolutionized the studies of translation. It was originally developed for yeast and mammalian cells in culture (Ingolia et al., 2009). Herein, we describe a plant-optimized hands-on ribosome footprinting protocol derived from previously published procedures of polysome isolation (Ingolia et al., 2009; Mustroph et al., 2009) and ribosome footprinting (Ingolia et al., 2009; Ingolia et al., 2013). With this protocol, we have been able to successfully isolate and analyze high-quality ribosomal footprints from different stages of in vitro grown Arabidopsis thaliana plants (dark-grown seedlings [Merchante et al., 2015] and 13-day-old plantlets in plates and plants grown in liquid culture [unpublished results]).

[Background] The central role of translation in modulating gene activity has long been recognized, yet the systematic exploration of quantitative changes in translation at a genome-wide scale in response to a specific stimulus has only recently become technically feasible. The ribosome footprinting technology (often known as the Ribo-seq), developed originally for yeast and mammalian cells in culture, has revolutionized the studies of translation regulation and gene expression, as it allows to determine the exact positions of the ribosomes at a genome-wide scale and at a single-codon resolution (Ingolia et al., 2009).
  Prior to the development of Ribo-seq, the most common methods employed to study translation regulation in plants were the isolation of polysomal RNA via sucrose gradient centrifugation or translating ribosome affinity purification (TRAP) followed by Northern blotting, qRT-PCR, microarrays, or RNA-seq. The first method, known as polysome profiling, relies on resolving distinct polysomal fractions on a sucrose gradient via ultracentrifugation (Branco-Price et al., 2008; Missra and von Arnim, 2014; Li et al., 2015). By comparing different plant growth conditions or mutants, one could infer the changes in the rates of translation from observing a shift in the distribution of mRNAs between polysomal fractions. For example, if a transcript becomes more abundant in the monosomal fraction with the concomitant decrease in the higher order polysomes, the translation of this mRNA is considered as down-regulated. The key limitation of this technique, however, is its low resolution of higher-order polysomes (and thus mild quantitative changes in translation are often missed) and the inability to differentiate between polysomal RNAs that undergo active translation versus are loaded with arrested ribosomes (for example, those ‘stuck’ in the upstream open reading frames of a transcript). The second polysomal RNA isolation technique, TRAP, is based on the stable expression of an epitope-tagged ribosomal protein followed by the immunoprecipitation of entire ribosomes along with their associated mRNAs (Zanneti et al., 2005; Reynoso et al., 2015). While this latter method accommodates both global and tissue-specific studies of translation (achieved by driving the expression of a tagged ribosomal protein in a ubiquitous versus tissue-specific manner), its use is limited to transformable species where transgenic lines can be generated. Furthermore, transcriptomic analysis of TRAP samples per se does not provide a quantitative measure of translation (unless coupled with Ribo-seq [Juntawong et al., 2014]), as any mRNA with one or more ribosomes bound to it will be purified by TRAP. Also, since TRAP relies on epitope-tagging and the tag may interfere with the function of the ribosome, the regulation of translation of some mRNAs may be disrupted in the TRAP transgenic lines, e.g., due to a reduced ability of the tagged ribosome to associate with specific proteins at certain stages of translation. Another limitation of TRAP is that it typically uses a specific redundant isoform of a ribosomal protein for tagging, such as RPL18, and thus likely purifies only a subset of ribosomes that carry just that RPL18 variant. Given that there are multiple RPL18-like proteins in plant genomes, using one specific ribosomal protein isoform for tagging misses the ribosomes that utilize an alternative RPL18 isoform.
  The method of choice for our studies, the Ribo-seq, does not involve transgenic line generation nor affinity purification, thus avoiding many of the limitations of the aforementioned earlier techniques. Most importantly, the single-codon resolution of the ribosome footprinting technology allows researchers to map the ribosomes on the mRNAs and thus clearly distinguish between the transcripts harboring productive ribosomes translating the main genic open reading frames versus transcripts associated with non-productive ribosomes arrested in the 5’UTRs. Not only does this method offer a snapshot of a whole-genome view of ribosomal distribution at an unprecedented resolution, it also enables the true quantitative measure of translational efficiency of every expressed gene in the genome by correlating the Ribo-seq data with the transcriptional information obtained via RNA-seq. Nonetheless, even the Ribo-Seq has its own drawbacks, as it cannot discriminate between mRNA subpopulations with different translation efficiencies, giving an average translation efficiency readout for each expressed gene.
  Herein, we provide a plant-optimized Ribo-seq protocol that enables the study of translation regulation through the isolation of high-quality ribosomal footprints from different developmental stages of in vitro grown Arabidopsis thaliana seedlings. It describes step by step how to pellet and digest polysomes, isolate monosomes, extract the mRNA footprints, and generate sequencing libraries for the Illumina platform. The protocol also describes the preparation of parallel RNA-seq libraries to account for transcriptional regulation. We conclude the description of our method with a brief summary of how to analyze the sequencing results.

[Abstract]

Materials and Reagents

  1. Materials
    1. Miracloth
    2. 30 ml NalgeneTM High-Speed polycarbonate centrifuge tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3118-0030 ) (or equivalent) to centrifuge the plant extracts
    3. 5 ml polypropylene thin-wall ultracentrifuge tubes (Beckman Coulter, catalog number: 326819 )
    4. 10 ml ultracentrifuge 14 x 89 mm tubes with isopycnic caps (BioComp Instruments, catalog number: 105-914A )
    5. Fine scale
    6. 10 ml syringe
    7. Cannula to be attached to the syringe (Thomas Scientific, catalog number: 1193G13 )
    8. Pre-sterilized, RNase-free 5 ml, 1 ml, 200 µl and 10 µl micropipette tips
    9. Pre-sterilized, RNase-free 2 ml and 1.5 ml microcentrifuge tubes
    10. 15 and 50 ml Falcon tubes
    11. Stoppers to secure the lids of 1.5 ml tubes
    12. Razor blades
    13. Dynabeads mRNA Purification Kit (Thermo Fisher Scientific, AmbionTM, catalog number: 610-06 )
    14. 2 ml microcentrifuge tubes
    15. 1.5 ml non-stick, RNase-free microtubes (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: AM12450 )
    16. MyOneTM Streptavidin C1 DynaBeads® (Thermo Fisher Scientific, InvitrogenTM, catalog number: 65601 )
    17. 200 μl PCR strip tubes

  2. Reagents
    1. Liquid nitrogen
    2. RNase-free, sterile, MilliQ water
    3. Tris base
    4. Sucrose
    5. Potassium chloride (KCl)
    6. Sodium chloride (NaCl)
    7. Magnesium chloride (MgCl2)
    8. Ethyleneglycol-bis(2-aminoethylether)-N,N,N’,N-tetraacetic acid (EGTA)
    9. Ethylene diamintetracetic acid (EDTA)
    10. Dithiothreitol (DTT)
    11. Brij-35 [Polyoxyethylene(23)lauryl ether]
    12. Triton X-100
    13. Igepal CA 630 (Octylphenyl-polyethylene glycol)
    14. Tween 20 (Polyoxyethylene sorbitan monolaurate 20)
    15. Cycloheximide (see Note 1)
    16. Chloramphenicol (see Note 1)
    17. Lithium chloride (LiCl)
    18. Sodium acetate (NaOAc)
    19. Sodium bicarbonate (NaHCO3)
    20. Sodium carbonate (Na2CO3)
    21. Sodium dodecyl sulfate (SDS)
    22. Sodium hydroxide (NaOH)
    23. Trisodium citrate (Na3C6H5O7)
    24. Dimethylsulfoxide (DMSO), PCR grade
    25. Ethanol, molecular biology grade
    26. Water-saturated, acid phenol, molecular biology grade (see Note 2)
    27. Chloroform, molecular biology grade (see Note 2)
    28. Isoamyalcohol
    29. Isopropanol, molecular biology grade
    30. PEG8000
    31. 15 mg/ml GlycoBlueTM (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9515 )
    32. 10 bp DNA ladder (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10821015 )
    33. 2x denaturing sample buffer with dye (Thermo Fisher Scientific, InvitrogenTM, catalog number: LC6876 ) (see Note 3)
    34. SYBR® Gold, 10,000x in DMSO (Thermo Fisher Scientific, InvitrogenTM, catalog number: S11494 ) (see Note 4)
    35. dNTP mix, 10 mM (Thermo Fisher Scientific, InvitrogenTM, catalog number: 18427-013 )
    36. OmniPur® polyethylene glycol 8000 (EMD Millipore, Calbiochem®, catalog number: 6510 )
    37. Universal miRNA cloning linker (New England Biolabs, catalog number: S1315S )
    38. Boric acid
    39. Acetic acid
    40. 12-well 15% polyacrylamide TBE-Urea gels (Bio-Rad Laboratories, catalog number: 4566055 ) (see Note 5)
    41. 30% acrylamide/bisacrylamide (29:1) (Bio-Rad Laboratories, catalog number: 161-0156 ) (see Note 5)
    42. Ammonium persulfate (APS)
    43. TEMED (N,N,N’,N’-Tetramethylethane-1,2-diamine) (Sigma-Aldrich, catalog number: T7024 )
    44. Glycerol
    45. Bromophenol blue

  3. Enzymes
    1. SUPERase-InTM RNase inhibitor (Thermo Fisher Scientific, AmbionTM, catalog number: AM2694 )
    2. TURBOTM DNase (2 U/μl) (Thermo Fisher Scientific, AmbionTM, catalog number: AM2238 )
    3. RNase I (100 U/μl) (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM2294 )
    4. SuperScript® III reverse transcriptase (Thermo Fisher Scientific, InvitrogenTM, catalog number: 18080-093 )
    5. T4 polynucleotide kinase, T4 PNK4 (New England Biolabs, catalog number: M0201 ), supplied with 10x T4 PNK buffer (New England Biolabs, catalog number: B0201 ) (see Note 6)
    6. T4 RNA ligase 2, truncated (New England Biolabs, catalog number: M0242 ), supplied with PEG 8000 50% and 10x T4 Rnl2 buffer
    7. Phusion high-fidelity DNA polymerase (2 U/μl) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: F-530S )
    8. CircLigaseTM ssDNA ligase (100 U/μl) (Epicentre, catalog number: CL4115K )

  4. Oligonucleotides
    1. Reverse transcription primer for split-adapter circularization (see Note 7)
      NI-NI-9: [P]AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGC[SC18]CACTCA[SC18]TTCAGACGTGTGCTCTTCCGATCTATTGATGGTGCCTACAG (Ingolia et al., 2009)
    2. Size marker oligos (Ingolia et al., 2009) (see Note 8)
      oNTI265: rArUrGrUrArCrArCrGrGrArGrUrCrGrArGrCrUrCrArArCrCrCrGrCrArArCrGrCrGrA
      oNTI268: rArUrGrUrArCrArCrGrGrArGrArCrCrCrGrCrArArCrGrCrGrA
    3. rRNA subtraction oligos (all the oligos here have a 5’ TEG-linked biotin and were obtained from IDT),These oligos are based on the most abundant rRNA sequences and transposons that were found using 3-day-old Arabidopsis seedlings and the ribosome footprinting protocol described herein (see Note 9)
      rRNABio1: 5’-gataaccgtagtaattctagag-3’
      rRNABio2&4: 5’-TGATTCATGATAACTCGACGGACGACGCGGATTACGGTGGCGGC-3’
      rRNABio5&3: 5’-GTCGCTGCCGTGATCGTGGTCTCCATCGAGTCTTTGAACGCAAG-3’
      Bio-Tranpos: 5’-GAGGGATGCAACACGAGGAGTTCCCGGGAGGTCA-3’
      Bio-5S: 5’-AAGCCTTCTGGCCGAGGGCACGTCTGCCTGGGTGTCACAA-3’
      Bio-18S: 5’-AAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTG-3’
    4. Amplification primers
      NI-NI-2: 5’-AATGATACGGCGACCACCGAGATCTACAC-3’ (Ingolia et al., 2009)
      NI-NI-3: 5’-CAAGCAGAAGACGGCATACGAGATAGTCGTGTGACTGGAGTTCAGACGTGTGCTCTTCCG-3’ (Ingolia et al., 2009)
      Alternative indexed primers
      Index 1:
      5’-CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCG-3’
      Index 2:
      5’-CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCAGACGTGTGCTCTTCCG-3’
      Index 3:
      5’-CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGACGTGTGCTCTTCCG-3’
      Index 4:
      5’-CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTCAGACGTGTGCTCTTCCG-3’
      Index 5:
      5’-CAAGCAGAAGACGGCATACGAGATCACTGTGTGACTGGAGTTCAGACGTGTGCTCTTCCG-3’
      Index 6:
      5’-CAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAGTTCAGACGTGTGCTCTTCCG-3’
      Index 7:
      5’-CAAGCAGAAGACGGCATACGAGATGATCTGGTGACTGGAGTTCAGACGTGTGCTCTTCCG-3’

  5. Solutions
    All solutions should be prepared with RNase-free, MilliQ water. Unless otherwise stated, all of them must be sterilized by autoclaving for at least 20 min and stored at room temperature.
    1. 1 M Tris-HCl, pH 7.0 (see Note 10)
    2. 1 M Tris-HCl, pH 8.0 (see Note 10)
    3. 1 M Tris-HCl, pH 9.0 (see Note 10)
    4. 1 M sucrose, prepared from Molecular Biology grade sucrose (autoclave for 10 min)
    5. 1 M KCl
    6. 5 M NaCl
    7. 1 M MgCl2
    8. 0.5 M EGTA, pH 8.0 (see Note 11)
    9. 0.5 M EDTA, pH 8.0 (see Note 11)
    10. 2 mM EDTA, 100 mM Na2CO3
    11. 2 mM EDTA, 100 mM NaHCO3
    12. 3 M NaOAc, pH 5.5
    13. 10% SDS (w/v)
    14. 1 M NaOH (no sterilization required)
    15. 4 M LiCl (filter-sterilize, do not autoclave)
    16. 1 M DTT (do not autoclave; aliquot and store at -20 °C)
    17. 100 mg/ml cycloheximide, prepared in DMSO (filter-sterilize; aliquot and store at -20 °C)
    18. 50 mg/ml chloramphenicol, prepared in ethanol (filter-sterilize; aliquot and store at -20 °C)
    19. Detergent mix 20% (w/v or v/v) of each of four detergents in water (Brij-35, Triton X-100, Igepal CA 630 and Tween 20)
    20. 50% PEG8000 (w/v), prepared by combining dry PEG with water in a 50 ml conical and mixing with gentle shaking overnight. No sterilization is required. PEG is very hygroscopic; so start adding PEG to less than half of the final volume of MilliQ water. Then, after it is dissolved, add more water if needed. PEG used for ligation (step C3) should not be more than 1 month old.
    21. 10% APS (w/v), Filter-sterilize and aliquot in small volumes (e.g., 500 µl). Store at -20 °C. Aliquots can be thawed and re-frozen several times.

  6. Buffers (see Recipes)
    1. Polysome extraction buffer (PEB)
    2. Sucrose cushion A (SCA)
    3. Sucrose gradients solutions
    4. Polysome digestion buffer (PDB)
    5. Sucrose cushion B (SCB)
    6. Polysome resuspension buffer (PRB)
    7. Total RNA extraction buffer (TREB)
    8. Alkaline fragmentation buffer (2x)
    9. Alkaline fragmentation stop/precipitation solution
    10. TAE (Tris/acetate/EDTA buffer) (50x)
    11. TBE (Tris/borate/EDTA buffer) buffer (5x)
    12. RNA gel extraction buffer (GEB)
    13. DNA gel extraction buffer (STE)
    14. SSC (20x)
    15. Subtraction bind/wash buffer (2x)
    16. 8% non-denaturing polyacrylamide gel (12 ml)
    17. Non-denaturing loading dye (6x)

Equipment

  1. 7-9 cm diameter porcelain mortar and pestle
  2. Small (30-50 ml) glass beakers
  3. Refrigerated Beckman Avanty J-25 centrifuge and Beckman JA17 rotor (or equivalent)
  4. Ultracentrifuge Beckman L8-70M (or equivalent) with swinging bucket rotors for polysome pelleting (Beckman Coulter, model: SW55Ti ) and sucrose gradient centrifugation (Beckman Coulter, model: SW41Ti or Thermo Fisher Scientific, Thermo ScientificTM, model: TH-641 ).
  5. Automatic P1000, P200 and P10 micropipettes
  6. Refrigerated tabletop microcentrifuge
  7. Orbital shaker
  8. Gradient Master Station, with both the gradient maker and the fractionation station (BioComp Instruments)
  9. Continuous UV light monitor (Bio-Rad Laboratories, model: EM-1 Econo UV Monitor )
  10. Fractionator (Gilson, model: FC203B )
  11. Nanodrop (or another spectrophotometer to quantify nucleic acid concentrations in small volumes of RNA)
  12. Nutator shaker
  13. Thermoblock
  14. Fume hood
  15. Mini-PROTEAN tetra cell polyacrylamide gel box (Bio-Rad Laboratories, catalog number: 165-8004 ) or equivalent and electrophoresis power supply
  16. UV transilluminator
  17. Vortex
  18. DynaMagTM-2 magnetic separation rack (Thermo Fisher Scientific, catalog number: 12321D )
  19. Thermocycler
  20. 2100 BioAnalyzer (Agilent Technologies, catalog number: G2940CA )
  21. HiSeq2000 (Illumina)

Procedure

The wet-lab protocol is divided into A, B, and C steps (see Figure 1 for an overview). A steps regard to the preparation of the ribosome footprints. B steps refer to the total mRNA isolation procedure. C steps represent the part of the protocol where the footprints and mRNA fragments are processed together to create the sequencing libraries. The order in which A and B steps are performed is up to the researcher.
Tissues need to be flash-frozen after the desired treatment, ground to a very fine powder in liquid nitrogen, and then stored in -80 °C until use. 0.5 g of this pulverized tissue are required for the ribosome footprints and 0.5-1 g are needed to prepare the mRNA fragments. It is good practice to first isolate polysomes without including the digestion step to ensure that intact polysomes are being isolated before proceeding to the preparation of the footprint libraries. As with all RNA protocols, the tissues should not be allowed to thaw in the absence of buffer at any moment and all materials and solutions need to be RNase free. All the work should be performed on ice, unless otherwise stated.
These protocols allow for the generation of ready-to-sequence Ribo-seq and RNA-seq libraries from frozen ground tissues in 9 days. The entire protocol is organized in working days. At the end of the protocol, we include a Data analysis section that describes the pipeline that leads to the identification of genes regulated at the level of translation by the treatment or mutation of interest.


Figure 1. Overview of the ribosome footprinting procedure. A. Blue panel: steps that are specific to ribosome footprinting; B. Green panel: steps specific to mRNA library preparation; C. Yellow panel: steps that are common to both types of libraries. A, B and C steps are also marked in the written protocol.

  1. Steps: Ribosome footprinting
    Figure 2 shows a flow chart of the procedure comprising the A steps.


    Figure 2. Overview of the A steps of the protocol

Day 1

Note: Day 1 is a long day, around 14-18 h, dependent on the number of samples to be processed and the skills of the worker.
A1. Polysome isolation

  1. Switch ‘on’ the Beckman Avanty J-25 centrifuge and the L8-70M ultracentrifuge to allow them to cool down to 4 °C. Pre-chill the JA17 and SW55Ti rotors as well.
  2. Pre-chill on ice a clean porcelain mortar and pestle, a clean beaker with 3 layers of Miracloth (arranged as a funnel), and a Nalgene centrifuge tube per each sample to be analyzed.
  3. Place 0.5 g of pulverized frozen tissue in the pre-chilled mortar and add immediately 2 ml of ice-cold polysome extraction buffer (PEB). Mix the sample well with the pestle so that all the tissue is thawed in contact with the buffer.
  4. Let the sample stand on ice (with occasional gentle mixing with the pestle) for 10 min or until the rest of the samples are prepared.
  5. Take all the mixture with a 1 ml pipette or pour it into the beaker through the Miracloth filter to remove the biggest particles. Squeeze the filter to make sure that all of the supernatant is being collected in the beaker. Wash the mortar with 1 ml of new PEB, and pass it through the Miracloth. Squeeze again, and then, wash the Miracloth with 1 ml of clean PEB. Squeeze. Overall, a total of 4 ml of PEB is used for 0.5 g of tissue.
  6. Transfer the extract from the beaker to a Nalgene centrifuge tube making sure to collect as much extract as possible (see Note 12).
  7. Equalize the weight of the tubes with PEB before putting them in the Beckman Avanty J-25 centrifuge, so that the difference between opposite tubes is not bigger than 0.1 g.
  8. Centrifuge at 4 °C, at 7,728 x g, for 30 min.
  9. While the samples are spinning, prepare the sucrose cushion for polysome pelleting (steps A1.10 and A1.11).
  10. Pre-chill on ice the 5 ml ultracentrifuge tubes and buckets for the Beckman SW55Ti rotor.
  11. The sucrose cushion for polysome pelleting is made from sucrose cushion A (SCA) (see Recipes). SCA can be prepared in advance and stored in aliquots at -20 °C. If so, thaw on ice the amounts of SCA aliquots that are needed for the experiment taking into account the volume in which the aliquots were prepared (see Note 13) and that each ultracentrifuge tube will contain 0.5 ml of sucrose cushion and 4.5 ml of the plant extract (see Note 14).
  12. Once thawed, spin the SCA aliquots briefly in a tabletop microcentrifuge to collect the sucrose solution on the bottom of the tubes.
  13. Transfer the total needed volume of SCA with some excess to a new pre-chilled tube that fits the total volume of SCA that is required for the experiment (see Note 15). Homogenize the SCA solution by mixing carefully with the 1 ml pipette avoiding the formation of bubbles.
  14. To the SCA, add the DTT, cycloheximide and chloramphenicol to a final concentration of 5 mM, 50 μg/ml, and 50 μg/ml, respectively (see SCA in Recipes). Mix well with the pipette avoiding bubbles and transfer 0.5 ml of the cushion to each of the ultracentrifuge tubes.
  15. Once the centrifuge (step A1.8) is finished spinning, pour the supernatant into a clean, pre-chilled 15 ml tube (or any other type of tube larger than 4.5 ml) (see Note 16).
  16. Bring the volume of the extract to 4.5 ml with PEB and mix it carefully.
  17. Transfer the 4.5 ml extract to the ultracentrifuge tube on top of the sucrose cushion. A good way to do this is to take the extract with a 1 ml micropipette, place the tip perpendicular to the tube wall at the upper part of the tube, and let the extract slowly drip along the wall. This way the cushion will not be disturbed (Schematic representation in Figure 3A).


    Figure 3. Schematic representation of how to load the sucrose cushions (A) and the sucrose gradients (B). Red arrows indicate the angle of the pipette. 

  18. Equalize the weight of opposite tubes carefully with PEB using fine scales.
  19. Place ultracentrifuge tubes into the pre-chilled buckets and those into the pre-chilled SW55Ti rotor, making sure that the number on the bucket coincides with the position in the rotor.
  20. Centrifuge at 4 °C at 256,677 x g, for 3.5 h.
  21. While the samples are spinning, prepare the sucrose gradients (steps A1.22-A1.29) (see Note 17).
  22. To make the sucrose gradients and to fractionate polysomes, use the Gradient Master Station from BioComp Instruments, Seton 7030 10 ml ultracentrifuge tubes, rubber caps with a small hole that close the ultracentrifuge tubes, marker block and syringe with a cannula from BioComp Instruments and follow manufacturer’s recommendations.
  23. Prepare at room temperature the 10% and 50% sucrose gradients solutions (see Recipes). Each gradient will need 5 ml of each sucrose solution. Prepare the amount needed for the experiment with some excess.
  24. Mark the 10 ml ultracentrifuge tubes according to the marker block with a permanent marker (see Note 18).
  25. Fill the marked tubes with the 10% sucrose solution up to the mark (5 ml approx.).
  26. With the syringe and the cannula underlay 50% sucrose solution below the 10% one to fill the tube entirely (another 5 ml approx.).
  27. Gently insert the rubber cap into the top of the tube at an angle, so that the hole in the cap enters the liquid last allowing the air to scape.
  28. Put the tubes in the gradient station’s magnet bucket and place them on the Gradient Master Station. Program the station in the gradient position, with a rotation angle of 81.5°, at speed 16, for 1:58 min.
  29. Once the gradients are prepared, place them carefully at 4 °C until they are used.
  30. When the centrifuge run (step A1.20) is close to finishing, prepare the polysome digestion buffer (PDB) (see Recipes) without adding the DNase and RNase. Also have ice-cold MilliQ water, 10 ml per sample, ready to wash the polysome pellets.
  31. Once the polysome pelleting ultracentrifuge is finished spinning, remove all the supernatant and cushion by quickly and carefully pouring it down.
  32. Fill the tube with ice-cold MilliQ water and immediately pour it down. Repeat this washing step to remove all of the sucrose. The polysome pellet has a flat jelly-like appearance at the bottom of the tube (see Note 19).
  33. Briefly dry the pellets to remove most of the water by placing them upside down over clean absorbent paper for 30 sec-1 min.
  34. Resuspend the pellets in 500 μl of polysome digestion buffer (PDB) by pipetting carefully and transfer the samples to new clean 1.5 ml tubes.

A2. Polysome digestion and monosome pelleting

Polysome digestion is performed in a total volume of 4.5 ml. In our hands, this increase in the volume of digestion compared to other available footprinting protocols (Ingolia et al., 2009) improves significantly the quality of the obtained footprints. Digestion is performed in PDB. After digestion, the sample is pelleted again on a sucrose cushion before the fractionation step. When testing the protocol for the first time, it is recommended to keep one sample undigested to ensure that the polysomes are intact before the digestion step.

  1. Spin the 1.5 ml tubes with 500 μl of resuspended polysomes (step A1.34) for 30 sec at 20,800 x g and at 4 °C in a microfuge.
  2. Transfer the supernatant to a 15 ml Falcon tube pre-filled with 4 ml of PDB. Discard the pellet.
  3. Add 12 μl of TURBO DNase 2 U/μl and 10 μl of RNase I to each 4.5 ml sample for digestion (see Note 20).
  4. Incubate for 2 h at room temperature with gentle agitation in an orbital shaker.
  5. During the incubation, prepare the sucrose cushions for the next pelleting step. The cushions are prepared as described for the polysome pelleting step (steps A1.10-A1.14) but with sucrose cushion B instead (SCB) (see Recipes).
  6. When digestion time is over, add 7.5 μl of SUPERase-In RNase inhibitor to each 15 ml Falcon tube where polysome digestion is taking place and mix well by inversion. Immediately load the digested extracts onto the sucrose cushions as in step A1.17. Weight the tubes and equalize the weight of opposite tubes carefully with PDB using fine scales.
  7. Centrifuge, wash and dry the monosome pellets as before (steps A1.20 and A1.31-A1.33).
  8. Resuspend the pellets by pipetting carefully in 300 μl of polysome resuspension buffer (PRB) (see Recipes).
  9. Transfer to a new clean 1.5 ml tube. Spin briefly, and transfer the supernatant to a new tube. Discard the pellet.

A3. Polysome fractionation

At this step, the digested monosomal fraction is separated from the rest of the extract. To profile the polysomes, we recommend the use of the Gradient Master Station. Have the ultracentrifuge rotor and its centrifuge buckets pre-chilled at 4 °C. The ultracentrifuge should already be cold from the pelleting step, so keep it cold.

  1. Take the tubes with the sucrose density gradients (steps A1.22-A1.29) out of the fridge and place them on ice.
  2. Load the 300 μl of resuspended digested polysome pellets (step A2.9) on top of the sucrose density gradients. To do this, place the pipette tip at the wall of the tube, at the upper limit of the 10% sucrose meniscus. With the tip positioned there, slightly elevate the solution without breaking the surface tension and keep the tip in contact with the wall (Schematic representation in Figure 3B). Very carefully load the sample on top of the gradient. The extract should expand on top of the sucrose (this can be seen) without entering in it (see Note 21).
  3. Equalize the weight of all tubes very carefully with PRB using fine scales (be careful not to disturb the gradients).
  4. Carefully insert the tubes in the pre-chilled Sorvall TH-641 buckets and close them well. Put the buckets in the pre-chilled rotor making sure that the number on the bucket coincides with the position in the rotor.
  5. Spin the gradients in the ultracentrifuge at 4 °C at 234,479 x g for 2.5 h.
  6. 5-10 min before the centrifugation is finished, switch ‘on’ the Gradient Master Station and prepare it for the fractionation steps (follow manufacturer’s instructions).
  7. Retrieve the rotor buckets and keep the tubes on ice until fractionation.
  8. Fractionate the sucrose gradients on the gradient station at 0.2 mm/sec following manufacturer’s indications. Measure the A260 of the collected material using the continuous UV monitor to identify the 80S ribosome peak.
  9. Analyze the undigested sample first to ensure that it contains intact polysomes and to determine the approximate location of the 80S ribosome peak.
  10. Then, fractionate the digested sample, which should have a much larger 80S monosome peak than the undigested sample. The number of fractions depends on the researcher’s needs. Typically, collecting in 20 fractions allows the monosome peak to fall in a maximum of 2 fractions. Collected this way, each fraction will contain an approximate volume of 0.5 ml. To collect the fractions, use 2 ml tubes.
  11. Keep the fractions(s) corresponding to the 80S peak and store the samples at -80 °C until the researcher is ready to isolate the footprints.
    A comparison between undigested and digested polysome profiles is shown in Figure 4.


    Figure 4. Undigested (A) and digested polysome (B) profiles

Day 2

A4. Footprint fragment purification

Purify RNA from the monosome fraction using the SDS/phenol method. Work in a fume hood and use a lab coat, gloves and goggles to prevent skin damage if phenol spills.

  1. Thaw the monosome fractions (step A3.11) on ice. Spin briefly to collect the sample at the bottom of the tube.
  2. Add 10% SDS to the monosome fraction to a final concentration of 1% (w/v) (e.g., 50 µl of SDS are needed if the sample was fractionated into 20 fractions of 500 µl each), mix the sample by vortexing and place it at 65 °C. Secure the lids.
  3. Heat acid phenol-chloroform (5:1) to 65 °C in a fume hood.
  4. Add 1 volume of hot acid phenol-chloroform to the monosome fraction, mix well by vortexing and incubate for 5 min at 65 °C. Keep vortexing frequently while processing all of the samples (see Note 22).
  5. Place samples on ice for 5 min, and then spin at 20,800 x g in a tabletop microcentrifuge 2 min at room temperature.
  6. Transfer the upper aqueous phase (the lower phase if phenol-chloroform) to a new 1.5 ml tube (see Note 23).
  7. Add 1 volume of acid phenol-chloroform and keep it for 5 min at room temperature vortexing frequently.
  8. Spin for 2 min at room temperature at 20,800 x g in a tabletop microcentrifuge.
  9. Carefully transfer the aqueous phase to a new 1.5 ml tube and add 1 volume of chloroform-isoamylalcohol (24:1). Vortex for 1 min at room temperature.
  10. Spin for 1 min at room temperature at 20,800 x g in a tabletop microcentrifuge.
  11. Transfer the aqueous phase to a new pre-chilled 1.5 ml tube. If the digested polysomes were fractionated into 20 fractions of 0.5 ml, a recovery of 400 μl aqueous phase is expected.
  12. Precipitate RNA using GlycoBlue as a coprecipitant. To 400 μl RNA add 45 μl of 3 M NaOAc (pH 5.5), and 6.75 μl of GlycoBlue. Mix well by inversion and add 675 μl of ice-cold isopropanol. Mix well by inverting the tube.
  13. Incubate at least 30 min at -80 °C or dry ice, and centrifuge for 30 min at 20,800 x g at 4 °C in a tabletop microcentrifuge.
  14. Discard the supernatant by inverting the tube, spin briefly to collect the drops, remove them with the pipette, and air-dry the pellet for 1 min at room temperature.
  15. Resuspend the RNA in 10 μl of 10 mM Tris-HCl, pH 8.0.
  16. Keep the footprint RNA at -80 °C until the size selection step in gel (see step C1), which will be performed in parallel with the fragmented mRNA samples.

Day 3
  1. Steps: total mRNA preparation
    B1. Total RNA extraction
    Multiple RNA purification protocols could be used at this step. The following is based on the Reuber and Ausubel RNA extraction protocol (Reuber and Ausubel, 1996) (see Note 24).
  1. Switch ‘on’ a Beckman Avanty J-25 centrifuge to cool it down to 4 °C. Pre-chill the polycarbonate Nalgene centrifuge tubes on ice and the JA-17 Beckman rotor in the refrigerator.
  2. Add 5 ml of ice-cold total RNA extraction buffer (TREB) (see Recipes) to the pre-chilled centrifuge tubes, keep them on ice and add 0.5-1 g of pulverized frozen tissue.
  3. Vortex and invert the tube to make sure that all the tissue is in contact and thawed in the buffer. Keep the extracts on ice until the remaining samples are processed.
  4. Add 1 volume of acid phenol (see Note 25), vortex to uniformly mix the phenol and extract. Keep the samples on ice vortexing frequently while the remaining samples are processed.
  5. Spin samples at 4 °C at 7,728 x g for 10 min.
  6. Transfer the upper aqueous phase (the bottom phase is phenol) to a new polycarbonate centrifuge tube prefilled with 5 ml of acid phenol, vortex, and spin the samples in the centrifuge as in step B1.5.
  7. Transfer the upper aqueous phase to a new polycarbonate centrifuge tube prefilled with 5 ml of chloroform, vortex, and spin the samples at 4 °C, 7,728 x g for 10 min.
  8. Collect the supernatant and divide it into 10 pre-chilled 2 ml microcentrifuge tubes with 500 µl extract in each (see Note 26).
  9. To each tube add 0.1 volumes of 3 M NaOAc, pH 5.5 mix, and add 2.5 volumes of 100% ethanol (see Note 27).
  10. Mix samples by inversion. Place samples on ice for 5-10 min.
  11. Collect nucleic acids by centrifugation in a microfuge at top speed at 4 °C for 20 min (see Note 28).
  12. Discard supernatant, spin samples briefly and remove the remaining supernatant with the pipette. Resuspend each pellet in 50 μl of ice-cold MilliQ water. At this step, all of the aliquots coming from the same original sample can be combined again in a single 1.5 ml tube that will contain 500 μl sample. Do not proceed to the next step until the pellets are completely resuspended.
  13. Add 500 μl of 4 M LiCl, mix by inversion and let sit on ice for 30 min or longer (see Note 29).
  14. Collect the RNA by centrifugation in a microfuge at 4 °C at top speed for 30 min, and discard the DNA-containing supernatant.
  15. Wash the pellet with 500 μl ice cold 70% ethanol, centrifuge at top speed for 10 min, discard the supernatant, and air-dry the pellets for 10-15 min.
  16. Dissolve RNA in 50 μl of ice-cold RNase-free water.
  17. Quantify RNA yield in a spectrophotometer at 260 nm and check quality in a 1% agarose TAE gel.
  18. Keep the total RNA samples at -80 °C until used to purify mRNA.

Day 4

B2. Purification of the mRNA

Purify mRNA from total RNA using oligo-dT-coated magnetic beads from the Dynabeads mRNA Purification Kit essentially as described by the manufacturer.

  1. Prepare 220 μl of 1x binding buffer by diluting it from the 2x binding buffer stock provided with the Dynabeads Kit.
  2. Resuspend the magnetic beads by vortexing the vial and transfer 150 μl per sample to a 1.5 ml non-stick tube.
  3. Collect beads by placing the tube on the magnetic rack for 30 sec and carefully pipette away the storage buffer.
  4. Immediately resuspend beads in 100 μl of 1x binding buffer. Repeat this procedure to wash beads again in 1x binding buffer and leave them in binding buffer.
  5. Take 100-150 μg of total RNA and dilute to a final volume of 50 μl with RNase-free water.
  6. Add 50 μl of 2x binding buffer and denature the RNA for 2 min at 80 °C.
  7. Place the samples immediately on ice and add 1 μl of SUPERase-In.
  8. Remove carefully the binding buffer from the magnetic beads (step B2.4), add the entire RNA sample to the beads and resuspend carefully with the pipette.
  9. Incubate for 5 min at room temperature with gentle agitation in an orbital shaker to allow mRNA to bind to the beads.
  10. Return the tube to the magnetic rack to collect the beads for 30 sec and pipette away the unbound sample.
  11. Wash the beads twice with 100 μl of wash buffer B provided with the Dynabeads Kit.
  12. Remove all of the wash buffer B and resuspend the beads in 20 μl of 10 mM Tris-HCl, pH 7.0 mixing carefully with the pipette.
  13. Elute the RNA by heating the beads to 80 °C for 2 min.
  14. Immediately place the tube on the magnetic rack for 30 sec and transfer the eluted mRNA to a new tube.
  15. Save the mRNA at -80 °C if not going directly to the alkaline lysis step (step B3).

B3. Alkaline lysis

At this step the mRNA will be randomly fragmented and then, the bands with the same size as the footprints will be size-selected (C1). It is important to empirically determine the time of fragmentation, so that the sample is properly fragmented, yet the majority of the sample is not under 28 nt (see Figure 5A).

  1. Prepare the mRNA fragmentation reaction by mixing 20 μl of RNA (step B2.14) with 20 μl of 2x alkaline fragmentation buffer (see Recipes).
  2. Incubate the fragmentation reaction for 20-40 min at 95 °C. If possible, use a thermal cycle to keep temperature stable and to ensure reproducible conditions.
  3. Return the reaction to ice immediately.
  4. Pipette 560 μl of alkaline fragmentation stop/precipitation solution (see Recipes) into a non-stick microfuge tube on ice and add 40 μl of the fragmentation reaction to the stop solution. Mix by inverting the tube.
  5. Add 600 μl of ice-cold isopropanol and mix by inversion (see Note 30). Precipitate for 30 min at -80 °C or longer.
  6. Spin at 4 °C, at 20,800 x g for 30 min.
  7. Discard the supernatant, let the pellet air-dry for 1 min and resuspend in 10 μl 10 mM Tris-HCl, pH 8.0.

The fragmented mRNA obtained can be kept at -80 °C until gel size selection, which is performed side-by-side with the ribosome footprints samples.

  1. Steps: Sequencing library preparation
    C1. Size selection
    At this step, footprints and fragmented mRNA can be processed in parallel. As reference for size, specifically designed RNA primers of 25 and 34 nt (oNTI265 and oNTI268 [Ingolia et al., 2009]) should be run with the samples in parallel lanes and the bands delimited by these primers selected and cut out of the gel and saved.
  1. Pre-run a 12-well 15% TBE-Urea gel in 1x TBE (see Recipes) in a Mini-PROTEAN tetra cell polyacrylamide gel box at 200 V for 15 min.
  2. Prepare gel samples
    a. Add 5 μl of 2x denaturing sample buffer with dye to the 10 μl samples of footprint RNA and fragmented mRNA (from steps A4.15 and B3.7, respectively).
    b. Prepare the loading control by combining 0.5 μl of 10 bp ladder, 4.5 μl of water and 5 μl of 2x sample buffer
    c. Prepare the size-control RNA oligos: add 1 μl of each oligo (oNTI265 and oNTI268) diluted to 10 μM, 4 μl of MilliQ water and 5 μl of 2x sample buffer per gel lane (see Note 31).
  3. Keep the samples on ice until all of them are prepared.
  4. Denature samples, ladder and oligos at 80 °C for 90 sec.
  5. Load samples on the gel and run for 50-65 min at 200 V (see Note 32).
  6. Stain the gel with 1x SYBR Gold in 1x TBE by gentle shaking for 5 min.
  7. Visualize the gel in a UV transilluminator. In Figure 5A, a gel image shows how footprints and fragmented RNA libraries should look like.
  8. Excise the 25-34 nt region (demarcated by size marker oligos) from the footprinting and mRNA samples. Excise the oligos as well (combining both bands together) to use as a control for RNA recovery. It is also useful to perform a control of ligation to the polyadenylated linker with them (step C3).
  9. Extract RNA from gel slices
    1. Place the gel slice in a 1.5 ml non-stick RNase-free microfuge tube.
    2. Add 400 μl of RNA gel extraction buffer (GEB) (see Recipes).
    3. Freeze for 15-30 min at -80 °C or on dry ice.
    4. Place on a room temperature nutator overnight.

Day 5
  1. Spin for 1 min at maximum speed in a microfuge and recover the eluate to a new tube (see Note 33). 
  1. Precipitate RNA from gel extraction
    1. Take 400 μl of the eluate, add 1.5 μl of GlycoBlue, mix by inversion, and add 500 μl of isopropanol.
    2. Precipitate at least for 30 min at -80 °C or on dry ice.
    3. Spin at 4 °C at 20,800 x g for 30 min.
    4. Decant supernatant carefully and then spin briefly to collect the supernatant at the bottom of the tube. Remove the residual liquid with the micropipette.
    5. Air-dry the pellet for 1 min at room temperature.
  2. Resuspend the RNA in 10 μl of 10 mM Tris-HCl, pH 8.0 and transfer to a clean microfuge tube.

C2. Dephosphorylation

This dephosphorylation step is necessary to allow ligation of the 24-35 nt RNA fragments to the Universal miRNA linker.

  1. Add 33 μl of ice-cold MilliQ water to the 10 μl RNA (step C1.11).
  2. Denature for 90 sec at 70 °C and then equilibrate at 37 °C.
  3. Add 5 μl of 10x polynucleotide kinase buffer, 1 μl of SUPERase-In and 1 μl of T4 polynucleotide kinase and mix well by pipetting.
  4. Incubate for 1 h at 37 °C and heat-inactivate for 10 min at 70 °C.
  5. Precipitate RNA by adding 39 μl of water, 1 μl of GlycoBlue, 10 μl of 3 M NaOAc, mix well by inverting the tube, and then add 150 μl of ice-cold isopropanol. Mix well by inverting the tube. Incubate at least 30 min at -80 °C or on dry ice.
  6. Pellet RNA, remove liquid, and air-dry as above (steps C1.10b-C1.10e)
  7. Resuspend the dephosphorylated RNA in 5 μl of 10 mM Tris-HCl, pH 8.0 and transfer it to a clean microfuge tube.

C3. Linker ligation

The ligation reaction requires a high concentration of the preadenylylated linker (Universal miRNA cloning linker). The preadenylated primer should be diluted to 205 ng/μl with 5 μl of Tris-HCl, pH 8.0. It is useful to monitor the extent of ligation, especially in preliminary experiments. For this, a ligation reaction can be set with the primers that have been recovered from the gel (step C1.8). This also gives an idea of how well gel recovery worked.

  1. Prepare the ligation reaction mix with excess (see Note 34). For each reaction, mix 2 μl of 10x T4 Rnl2 buffer, 1 μl of SUPERase-In, and 6 μl of PEG 8000 50% (w/v) (the PEG 8000 should be no more than a month old).
  2. Combine on ice the RNA sample (from step C2.7), 2 μl of Universal miRNA Linker 205 ng/μl and MilliQ water to a final volume of 10 μl.
  3. Denature for 2 min at 80 °C and then equilibrate to 37 °C.
  4. Add 9 μl of reaction mix from step C3.1 to each sample and mix well.
  5. Add 1 μl of T4 Rnl2(tr) to each sample and mix well.
  6. Incubate for a minimum of 2.5 h at room temperature (see Note 35).

Day 6
Note: Can be day 5 if a 2.5 h ligation is performed instead of letting it sit overnight. Herein, day counting continues as if an overnight incubation was done.

  1. Stop ligation and precipitate RNA by adding 40 μl of 3 M NaOAc, pH 5.5, 1.5 of μl GlycoBlue, and 338 μl of MilliQ water. Mix well and then add 500 μl of isopropanol. Mix well by inverting the tube.
  2. Incubate at -80 °C, pellet, remove supernatant, and air-dry as described above (steps C1.9 and C1.10).
  3. Resuspend ligation products in 5 μl of 10 mM Tris-HCl, pH 8.0.

C4. Gel purification of the ligated products

This gel purification is used to separate ligation products from the pre-adenylated linker, which is only 17 nt long.

  1. Pre-run a 12-well 15% TBE-Urea gel in 1x TBE at 200 V for 15 min.
  2. Prepare gel samples
    1. Add 5 μl of 2x denaturing sample buffer to the 5 μl RNA samples (from step C3.9).
    2. Prepare the 10 bp ladder and the size control oligos (oNTI265 and oNTI268) as in steps C1.2b and C1.2c.
  3. Keep the samples on ice until all of them are prepared.
  4. Denature samples, ladder and oligos at 80 °C for 90 sec.
  5. Load samples on gel and run for 50-65 min at 200 V (see Note 36).
  6. Stain the gel for 5 min in 1x TBE, 1x SYBR Gold while shaking gently in an orbital shaker.
  7. Visualize the gel in a UV transilluminator and excise the desired bands, which will now be 17 nt longer than the input. Figure 5B shows an image of the ligated samples.
  8. Extract RNA from the gel slices as described above (step C1.9).

Day 7
  1. Precipitate RNA from the gel extraction as described above (step C1.10).
  2. Resuspend purified ligation product in 10 μl of 10 mM Tris-HCl, pH 8.0.

C5. Reverse transcription

The reverse transcription (RT) is performed with the SuperScript III Reverse Transcriptase Kit from Invitrogen. RT primer NI-NI-9 (Ingolia et al., 2009) should be present in modest excess of target RNA (see Note 37).

  1. Prepare at room temperature a reaction mix containing (per reaction) 4 μl of 5x first-strand buffer, 1 μl of MilliQ water, 1 μl of dNTPs (10 mM each), 1 μl of 100 mM DTT, 1 μl of SUPERase-In and 1 μl SuperScript III. Allow some excess for the mix.
  2. Add 1 μl of the 1.25 μM primer NI-NI-9 to the 10 μl ligated RNA (from step C4.9).
  3. Denature the RNA and primer for 2 min at 80 °C, then place reactions on ice.
  4. Add 9 μl of the RT reaction mix and mix well.
  5. Incubate for 30 min at 48 °C.

C6. Removal of RNA template

  1. Add 2.2 μl of 1 N NaOH to each reaction. Mix well.
  2. Incubate at 98 °C for 20 min.
  3. Neutralize the reaction by adding 20 μl of 3 M NaOAc, pH 5.5, 1.5 of μl GlycoBlue, and 156 μl of MilliQ water. Mix well by inverting the tube.
  4. Immediately add 300 μl of isopropanol and mix well by inverting the tube.
  5. Precipitate and pellet cDNA, remove the supernatant, and air-dry pellets as described above for RNA (steps C1.10b-C1.10e).
  6. Resuspend the cDNA pellet in 5 μl of 10 mM Tris-HCl, pH 8.0.

C7. Gel purification of product

This gel purification step is necessary to separate the RT primer, which runs around at 100 nt, from the extended products, which may be as short as 128 nt. The poor separation of these bands is the reason that a large excess of primer causes background.

  1. Pre-run a 12-well 15% TBE-Urea gel in 1x TBE at 200 V for 15 min.
  2. Prepare gel samples: add 5 μl of 2x denaturing sample buffer to the 5 μl of cDNA from the RT (step C6.6). Prepare the 10 bp ladder as described before (step C1.2b). Prepare the size control by combining 2 μl of unextended 1.25 μM NI-NI-9 primer with 4.5 μl of water and 5 μl of 2x denaturing sample buffer.
  3. Denature samples at 98 °C for 90 sec.
  4. Load samples on gel and run for 50-60 min at 200 V.
  5. Stain the gel for 5 min in 1x TBE, 1x SYBR Gold on a gentle shaker.
  6. Visualize the gel and excise the extended RT product bands on a UV transilluminator. Figure 5C shows the gel image of reverse-transcribed samples and the NI-NI-9 oligo control.
  7. Extract DNA from the gel slices as described above (step C1.9), but use DNA gel extraction buffer (STE) (see Recipes), instead of GEB.

Day 8
  1. Precipitate DNA from the gel extraction as described above for RNA (step C1.10).
  2. Resuspend reverse transcription products in 15 μl of 10 mM Tris-HCl, pH 8.0.

C8. Circularization

For the circularization step, the CircLigase ssDNA ligase from Epicentre is used.

  1. Prepare a reaction mix with some excess. For one reaction, use 2 μl of 10x CircLigase buffer, 1 μl of 1 mM ATP, 1 μl of 50 mM MnCl2 and 1 μl of CircLigase, and mix well.
  2. Add 4 μl of reaction mix to the 15 μl of resuspended cDNA samples from step C7.9 and mix well.
  3. Incubate for 1 h at 60 °C.
  4. Heat-inactivate for 10 min at 80 °C.

C9. Subtractive hybridization

The products of the circularization reaction can be used directly as a template for PCR amplification of a complete sequencing library. For ribosome footprinting samples, the circularization reaction can instead be used as a direct input to subtractive hybridization to remove high-abundance rRNA-derived sequences. A first round of footprints sequencing can be performed to analyze which are the most abundant rRNA sequences, and with that information, design biotinylated oligos to remove those abundant rRNA sequences in the future experiments.

  1. Prepare the Subtraction Pool with 2 μl of each distinct biotinylated rRNA subtraction oligo from the 200 μM oligo stocks (see Materials and Reagents) and add water to a final volume of 40 μl. This pool can be stored at -20 °C indefinitely.
  2. Combine 10 μl of circularized libraries from step C8.4 with 2 μl of the Subtraction Pool, 2 μl of 20x SSC (see Recipes) and 6 μl of MilliQ water (see Note 38).
  3. Denature the samples at 100 °C for 90 sec and anneal by lowering the temperature to 37 °C at a ramp of 0.1 °C/sec in a thermal cycler. Keep at 37 °C for 15 min.
  4. Dilute 500 μl of 2x subtraction bind/wash buffer (see Recipes) with 500 μl of water to make 1 ml of 1x bind/wash buffer. Resuspend the Dynabeads (10 mg/ml Invitrogen) by vortexing and use 25 μl per subtraction reaction.
  5. Collect beads by placing the tube on the magnetic rack for 30 sec and carefully pipette away the storage buffer.
  6. Immediately resuspend beads in one volume of 1x bind/wash buffer. Repeat this procedure to wash beads 2 more times in 1x bind/wash buffer. Pipette the 1x bind/wash buffer away.
  7. Resuspend the washed beads in 2x bind/wash buffer, 20 μl per subtraction reaction, and equilibrate at 37 °C.
  8. Add the 20 μl sample (step C9.3) to 20 μl of washed Dynabeads and incubate for 15 min at 37 °C with shaking.
  9. Recover the 35 μl eluate and transfer it to a new 1.5 non-sticky tube.
  10. Add 3 μl of GlycoBlue, 12 μl of 5 M NaCl, and 148 μl of water and mix well by pipetting gently.
  11. Precipitate by adding 150 μl of isopropanol and mix by inverting the tube (see Note 39).
  12. Precipitate DNA as described above (steps C1.10b-C1.10e).
  13. Resuspend in 10 μl of 10 mM Tris-HCl, pH 8.0.

C10. PCR amplification

PCR amplification does not substantially distort the relative representation of different sequences in the library until the point at which the primers are nearly exhausted and, presumably, there is competition between sequences for primer binding as well as for re-annealing of template strands as opposed to hybridization. For this reason, it is useful to set up PCR reactions with different cycle numbers to optimize the yield for each individual template without saturating the reaction. If a large quantity of library is needed, this preliminary optimization can be used to select amplification conditions for a larger reaction.
Once these conditions are set up, it is recommended to perform multiple independent amplification reactions for each library rather than a single reaction in a larger volume (e.g., set up a 100 μl PCR mixture, distribute it in 5 different PCR tubes, run the PCRs and then combine the products again in a single tube). Since PCR amplification bias appears to be random, the bias in the amplification becomes less severe when multiple independent reactions are combined, so that the resulting sequencing reads are distributed more evenly along the transcripts. For this amplification step, use the Phusion High-Fidelity DNA Polymerase Kit.
To multiplex multiple libraries in one sequencing round, perform the amplification PCR with Indexed oligos that allow the de-convolution of the reads of individual libraries based on their barcodes included in the oligos (see Materials and Reagents).

  1. Prepare a 100 μl PCR mixture by combining 20 μl of 5x HF buffer, 2 μl of 10 mM dNTPs, 0.5 μl of 100 μM NI-NI-2 primer (Ingolia et al., 2009), 0.5 μl of 100 μM NI-NI-3 (or an alternative indexed primer: different combinations of NI-NI-2 and indexed primer should be used for all libraries to be multiplexed together in a single sequencing lane), 5 μl of the 10 μl sample as template (step C9.14), 71 μl of nuclease-free water, and 1 μl of Phusion polymerase. At least for 1 primer pair prepare a non-template negative control reaction.
  2. Split the 100 μl mixture into five 200 μl strip tubes, 20 μl per tube, for each footprinting and mRNA library.
  3. Perform PCR using the following program: 30 sec initial denaturation at 98 °C, followed by X cycles of 10 sec denaturation at 98 °C, 10 sec annealing at 65 °C, and 5 sec extension at 72 °C. When determining the proper number of cycles for the PCR amplification, remove strip tubes at the end of the extension phase after 8, 10, 12, 14 and 16 cycles. For the oligo control, remove the non-template control at 12 cycles. Allow one of these non-template oligo controls to be run per gel with PCR products. When visualizing the PCR amplification in the gel, choose the best number of cycles to amplify the libraries that gives the best amplification but does not saturate the reaction (see Note 40).
  4. Once the optimal number of cycles is established, perform the 5 independent PCRs per library with the selected conditions.

C11. Gel purification of the PCR products

  1. Prepare samples for gel purification of the PCR products: add 3.3 μl of 6x non-denaturing loading dye (see Recipes) to each PCR reaction. In parallel, prepare one ladder sample for each gel: combine 1 μl of 10 bp ladder, 15.7 μl of water, and 3.3 μl of 6x dye.
  2. Prepare a 12-well 8% polyacrylamide non-denaturing gel (see Recipes) in 1x TBE and pre-run the gel at 180 V for 15 min.
  3. When setting up the conditions, load the five PCR amplifications of one template at different cycle numbers in a series of adjacent wells. This will require 10 wells in one gel for a pair of samples. Also load a 10 bp ladder sample and a 10-cycle oligo amplification sample on each gel. Once the optimal PCR conditions are determined, combine the 5 independent PCRs per library in a single tube and run two wells with 20 μl of the combined PCRs each (see Note 41).
  4. Run the gel for 40 min at 180 V.
  5. Stain the gel for 5 min in 1x TBE, 1x SYBR Gold on a gentle shaker.
  6. Visualize the gel and excise the amplified PCR product bands from the reaction that should have given a strong product band as well as still has remaining, unconsumed oligos (see Figure 5D). Avoid the faint no-insert band that will appear beneath the library band for some samples.
  7. Extract DNA from the gel slice as described above (step C7.7).


    Figure 5. Gel images of different steps of the protocol. A. Size selection: The black box delimits the part of the gel to be cut out of the gel to purify the RNA; B. Gel after ligation; C. Gel after retrotranscription; D. Gel after final library amplification. RB: Ribosome footprints, P: 24-28 nt primers, TR: total mRNA fragments, LP: ligated primers as ligation control, RP: NINI-9, the primer for reverse transcription as size control.

Day 9
  1. Precipitate, pellet, and dry the recovered DNA (steps C1.10b-C1.10e).
  2. Resuspend in 15 μl 10 mM Tris-HCl, pH 8.0.

C12. Sequencing library quantitation by BioAnalyzer

This step depends on the selected platform for sequencing, but the instructions presented below are mostly general.

  1. Check the concentration and purity of the sequencing library using the BioAnalyzer high sensitivity DNA assay.
  2. Use 2 μl of each library with 8 μl of water for a 1:5 dilution of each sample.
  3. The quantitation results are reliable only if the diluted sample concentration does not exceed 500 pg/μl. If the DNA concentration is substantially higher than this, prepare a less concentrated sample by further dilution and determine the sample concentration.
  4. When using pooled and barcoded libraries, adjust concentrations of the individual libraries to be pooled and then add equal amounts of each library to the desired number of HiSeq lanes. The way the libraries are multiplexed depends on the researcher’s needs. In our case, we pooled complete experiments together (2 biological replicates of mock and treatment for both footprints and total mRNA). This way, 8 barcoded libraries were combined together into 7 Illumina HiSeq lanes, with each lane run with the same mixture of 8 libraries. We typically obtain about 150 million reads per lane and between 10% to 20% of these reads correspond to useful footprints (the remaining 80% to 90% are mostly ribosomal rRNA sequences).

Data analysis

The bioinformatic analysis described herein for the study of translation regulation using ribosome footprinting data follows the pipeline described in Merchante et al. (2015). The inputs for this pipeline are the de-multiplexed Ribo-seq and RNA-seq datasets (fastq-files) from Illumina HiSeq2000 platform, the TAIR 10 Arabidopsis genome sequence, and TAIR 10 Arabidopsis gene models (https://www.arabidopsis.org/).

  1. General data preprocessing
    1. Using FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/), remove adaptor sequences and trim low-quality regions.
    2. Align reads to the Arabidopsis thaliana genome sequence using Tophat2 (https://ccb.jhu.edu/software/tophat/index.shtml).
    3. Select uniquely mapping, sense-orientation reads in the size range of 20-40 nt, discard alignments with more than two mismatches, and create read count summaries of coding (CDS) regions.

  2. Assessment of periodicity for quality control
    Ribosome footprints, but not the RNA-seq reads, should show a strong 3 nt periodicity, which represents the codon-long step-wise movement of the ribosome along the mRNA (Ingolia et al., 2009; Merchante et al., 2015). So, the presence of this periodicity can be used as a control of the quality of the footprints. To avoid edge effects, restrict analysis to the genes with 5’ and 3’ UTRs longer than 70 nt and CDSs longer than 150 nt. To avoid ambiguities with respect to the location of reads along a transcript, restrict analysis to reads of a fixed length - typically a value between 28 to 32 nt, determined by visual inspection (C1 steps) - that map in sense orientation to only one transcript.
    1. Transform genomic read coordinates into transcript coordinates.
    2. Visualize read distribution in the vicinity of the CDS start: project the selected reads into the common coordinate system of a ‘metagene’. Bin the transcript coordinates of the read start points using their relative location with respect to the start of the main open reading frame: a read that starts at the beginning of the CDS falls into bin ‘0’. To visualize the read distribution around the end of the CDS, proceed analogously but bin with respect to the end of the main open reading frame.
    3. Normalize the histogram of read start positions by the total number of reads selected and draw a region around bin ‘0’.

  3. edgeR analysis of differential transcription and translation
    1. Discard genes with small read counts (rpkm value smaller than 3 in any of the samples) and perform TMM normalization (Robinson and Oshlack, 2010).
    2. Model the experiment by a 2-factorial design, where factor one is, for example, ethylene/control treatment and factor two is RNA-seq/Ribo-seq treatment. Use the generalized linear models approach (glm edgeR) of the Bioconductor package edgeR (Robinson et al., 2010) to select genes that, in this example, i) respond to ethylene on the RNA level, ii) respond to ethylene on the footprint level, and iii) respond differently to ethylene on the RNA-level and the footprint level. Determine the false discovery rate using Benjamini and Hochberg’s method (Benjamini and Hochberg, 1995).
     
  4. Analysis of changes in translational efficiency
    1. Following the definition of Ingolia and colleagues (Ingolia et al., 2009), compute translational efficiency (TE) as the fraction of Ribo-seq footprint rpkm value over RNA-seq rpkm value: TE = rpkm(foot)/rpkm(mRNA).
    2. Compute log2-transformed TE-values of ethylene-treated plants and subtract corresponding control values: ΔTE = log2 (TEethylene) - log2 (TEcontrol)
    3. Compute z-score of ΔTE: zΔTE= (ΔTE-E[ΔTE])/√Var(ΔTE). Determine the false discovery rate using the R package fdrtool (Strimmer, 2008).

Notes

  1. Cycloheximide and chloramphenicol are very toxic and harmful to the environment.
  2. Both phenol and chloroform are acute toxins. Handle them in a fume hood and wear proper protection.
  3. The 2x denaturing sample buffer with dye contains formamide, which is a reproductive toxin. Use proper protection when handling it.
  4. Nucleid acid stains are typically mutagenic, use proper protection when handling them.
  5. Both acrylamide and bisacrylamide are neurotoxins, use proper protection when handling them.
  6. Make sure not to confound the proper T4 PNK (M0201) with the T4 Polynucleotide Kinase 3’ phosphatase minus (M0236) as with the latter one the ligation step will not work.
  7. [P] represents 5’-phosphorylation and [SC18] represents a hexa-ethyleneglycol spacer.
  8. ‘r’ indicates that these are RNA oligonucleotides.
  9. These oligos may need to be re-designed if different treatments, growth conditions, developmental stages, footprinting protocols, etc., are used.
  10. This protocol requires Tris-HCl at pH 7.5, 7.8, 8.0, 8.4, 8.8 and 9.0, but all intermediate-concentration Tris-HCl buffers can be made out of the Tris-HCl pH7.0, 8.0 and 9.0 listed here.
  11. EGTA and EDTA only dissolve after adjusting the pH.
  12. There will be foam due to the use of detergents. Make sure to collect all the bubbles as well.
  13. When preparing the aliquots, allow some excess as the solution is very viscous. 1.2 ml aliquots work well for two samples.
  14. The SW55Ti rotor allows spinning 6 samples at the same time, so a maximum of 3 ml of SCA will be needed per centrifugation round.
  15. Such as a 5 ml ultracentrifuge tube or a 15 ml Falcon tube.
  16. This step is necessary because the debris pellet can easily get dislodged if supernatant is not transferred to a different tube rapidly at the end of the spin; failure to transfer the supernatant immediately will result in carryover of the debris and compromise ultracentrifugation.
  17. Sucrose gradients can also be prepared at the monosome pelleting centrifuge-step A2.7.
  18. Approximately half of the tube.
  19. The polysome pellet will be clear if using etiolated seedlings and dark green if photosynthetic tissues are employed.
  20. If there is a control tube that is not going to be digested, do not add the RNase I to it and keep it on ice until digestion time is over.
  21. Be extra-careful when doing this to not disturb the gradient.
  22. If using 1.5 ml tubes to heat the phenol:chloroform, do not allow the temperature to get higher than 65 °C and secure the lids, as, due to the pressure, the lids can open quite easily and produce dangerous spills, leading to the loss of precious samples.
  23. Phenol alone can also be used to extract the RNA, but if this is the case, the aqueous phase can be the lower one due to the high concentration of sucrose in the extract.
  24. Other standard RNA extraction methods (e.g., Trizol) compatible with high-throughput sequencing library construction and next-gen sequencing can also be used.
  25. Phenol:Chloroform:Isoamylalcohol (25:24:1) can also be used.
  26. JA17 tubes in the Beckman Avanty J-25 can also be used.
  27. If using 2 ml centrifuge tubes, add 500 μl of extract to different microtubes prefilled with 50 μl 3 M sodium acetate pH 5.2, mix and add 1,375 μl of ethanol.
  28. 34,895 x g if the Beckman centrifuge is used.
  29. At this step, samples can be left overnight at 4 °C if necessary.
  30. The stop solution already contains 300 mM salt, so no more salt is needed to precipitate the RNA.
  31. It is easier to select the size if the samples are surrounded by the oligos, so, run two oligo lanes, at the beginning and end of the gel.
  32. The time of the run has to be determined empirically depending on the running cell. It is a good practice to start running a test with the primers. As a reference, for the first run do not let the lower blue band from the denaturing sample buffer run out of the gel. Note the position of the marker primers with respect to the dye and adjust the future run times to the primer test.
  33. The gel piece does not disintegrate or change appearance.
  34. Allow a relatively high excess as the mix is very viscous and difficult to pipette.
  35. If the room-temperature incubation is followed by an overnight incubation at 4 °C, ligation efficiency increases.
  36. As a reference, the ligated samples typically run just above the higher blue band of the sample buffer.
  37. When the primer is present in vast excess, there is increased background from unextended primer that can, in some cases, dominate the actual library products. It is best to determine the right proportion empirically. 1-2 μl of NI-NI-9 primer at 1.25 μM should work fine. This can be tested in the gel run afterwards: the unextended primer should still be present after the RT step, but the extended libraries should not be fainter than the primer itself.
  38. The circularized libraries were resuspended in 20 µl and only 10 µl are used for the subtractive hybridization step. Keep the remaining 10 µl at -80 °C as a back-up.
  39. The buffer conditions in the subtractive hybridization (high salt as well as citrate, which chelates magnesium) are incompatible with PCR and thus precipitation is required after the subtraction step.
  40. Look for the cycle number that results in the best amplification without a significant loss of primers, so that there won’t be competition between sequences for the primers. The conditions are usually stable between experiments. Therefore, once the optimal conditions have been determined for a subset of samples, they also prove suitable for other samples from different experiments. In our case, the best amplification was achieved at 14 cycles, and further tests with additional samples confirmed 14 cycles as optimal, negating the need for further optimization.
  41. Keep the rest of the PCR as a back-up.

Recipes

  1. Polysome extraction buffer (PEB)
    110 mM Tris-HCl, pH 8.8
    100 mM sucrose
    100 mM KCl
    75 mM NaCl
    20 mM MgCl2
    12.5 mM EGTA
    3 mM DTT
    6.25 μl/ml detergent mix
    25 μl/ml Triton X-100
    37.5 μg/ml cycloheximide
    25 μg/ml chloramphenicol
    Prepare fresh before the extraction and keep on ice until use
  2. Sucrose cushion A (SCA)
    1.75 M sucrose
    400 mM Tris-HCl, pH 9.0
    200 mM KCl 
    5 mM EGTA
    35 mM MgCl2
    5 mM DTT
    50 μg/ml cycloheximide
    50 μg/ml chloramphenicol
    Prepare in advance (minus DTT and antibiotics). Autoclave no longer than 15 min, make 1.2 ml aliquots and store at -20 °C. Add the last three compounds fresh for each experiment before use.
  3. Sucrose gradients solutions
    50%/10% (w/v) sucrose
    40 mM Tris-HCl, pH 8.4
    20 mM KCl
    10 mM MgCl2
    50 μg/ml cycloheximide
    50 μg/ml chloramphenicol
    1 μg/ml SUPERase-In
    Prepare fresh on the day of the experiment
  4. Polysome digestion buffer (PDB)
    20 mM Tris-HCl, pH 7.8
    150 mM NaCl
    5 mM MgCl2
    50 μg/ml Triton X-100
    1 mM DTT
    100 μg/ml cycloheximide
    12 μl TURBO DNase 2 U/μl per 4.5 ml of digestion volume
    10 μl RNase I per 4.5 ml of digestion buffer
    Prepare on the day of the extraction and keep on ice 
  5. Sucrose cushion B (SCB)
    1.75 M sucrose
    20 mM Tris-HCl, pH 7.8
    150 mM NaCl
    5 mM MgCl2
    5 mM DTT
    50 μg/ml cycloheximide
    50 μg/ml chloramphenicol
    1 μl/ml SUPERase-In
    Prepare in advance (minus the last four ingredients) and autoclave no longer than 15 min. Make 1.2 ml aliquots and store at -20 °C. Add the last four compounds fresh for each experiment before use.
  6. Polysome resuspension buffer (PRB)
    200 mM Tris-HCl, pH 8.4
    200 mM KCl
    25 mM EGTA
    35 mM MgCl2
    50 μg/ml cycloheximide
    50 μg/ml chloramphenicol
    1 μl/ml SUPERase-In
    Prepare the day of the extraction and keep in ice
  7. Total RNA extraction buffer (TREB)
    200 mM Tris-HCl, pH 8.0
    400 mM LiCl
    25 mM EDTA
    1% SDS
    Prepare in advance (the solution keeps for several months) and store at room temperature
  8. Alkaline fragmentation buffer (2x)
    2 mM EDTA
    100 mM Na2CO3, pH 9.2
    This solution is prepared by mixing 15 parts of 100 mM Na2CO3 with 110 parts of 100 mM NaHCO3. It will equilibrate with gaseous CO2 and raise in pH over time. It can be stored in tightly capped, single-use aliquots at room temperature for over a year.
  9. Alkaline fragmentation stop/precipitation solution
    60 μl 3 M NaOAc
    2 μl GlycoBlue 15 mg/ml
    500 μl RNase-free water per reaction
    Prepare right before use
  10. TAE (Tris/acetate/EDTA buffer) (50x)
    2 M Tris base
    1 M acetic acid
    50 mM EDTA
  11. TBE (Tris/Borate/EDTA buffer) (5x)
    445 mM Tris base
    445 mM boric acid
    10 mM EDTA
    Dilute the concentrated stock buffer to 1x right before use.
  12. RNA gel extraction buffer (GEB)
    300 mM NaOAc, pH 5.5
    1 mM EDTA
    0.25% SDS
  13. DNA gel extraction buffer (STE)
    300 mM NaCl
    10 mM Tris-HCl, pH 8.0
    1 mM EDTA
  14. SSC (20x)
    3 M NaCl
    300 mM trisodium citrate (Na3C6H5O7)
    Adjusted to pH 7.0 with HCl
  15. Subtraction bind/wash buffer (2x)
    400 μl 5 M NaCl
    2 μl 0.5 M EDTA
    10 μl 1 M Tris-HCl, pH 7.5
    590 μl water
  16. 8% non-denaturing polyacrylamide gel (12 ml)
    6.3 ml MilliQ water
    3.2 ml 30% acrylamide/bisacrylamide
    2.4 ml of 5x TBE
    84 μl of 10% APS
    10 μl TEMED
  17. Non-denaturing loading dye (6x)
    60% (v/v) glycerol
    0.03% (w/v) bromophenol blue

Acknowledgments

This work was supported by NSF grants MCB 1158181 and 0519869 to J.M.A.; MCB 0923727 to J.M.A. and A.N.S.; IOS 1444561 to J.M.A., A.N.S., and S.H.; NCSU-RISF to S.H. and J.M.A.; and a Marie Curie COFUND U-Mobility postdoctoral fellowship to C.M. (co-funded by the University of Malaga and the EU 7FP GA N_246550). We are grateful to Nick Ingolia for his help with implementing and troubleshooting this protocol in plants.

References

  1. Branco-Price, C., Kaiser, K. A., Jang, C. J., Larive, C. K. and Bailey-Serres, J. (2008). Selective mRNA translation coordinates energetic and metabolic adjustments to cellular oxygen deprivation and reoxygenation in Arabidopsis thaliana. Plant J 56(5): 743-755. 
  2. Benjamini, Y. and Hochberg, Y. (1995). Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Statist. Soc. B 57: 125-133.
  3. Ingolia, N. T., Ghaemmaghami, S., Newman, J. R. and Weissman, J. S. (2009). Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324(5924): 218-223. 
  4. Ingolia, N. T., Brar, G. A., Rouskin, S., McGeachy, A. M. and Weissman, J. S. (2013). Genome-wide annotation and quantitation of translation by ribosome profiling. Curr Protoc Mol Biol Chapter 4: Unit 4 18. 
  5. Juntawong, P., Girke, T., Bazin, J. and Bailey-Serres, J. (2014). Translational dynamics revealed by genome-wide profiling of ribosome footprints in Arabidopsis. Proc Natl Acad Sci U S A 111(1): E203-212. 
  6. Li, R., Sun, R., Hicks, G. R. and Raikhel, N. V. (2015). Arabidopsis ribosomal proteins control vacuole trafficking and developmental programs through the regulation of lipid metabolism. Proc Natl Acad Sci U S A 112(1): E89-98.
  7. Mustroph, A., Juntawong, P. and Bailey-Serres, J. (2009). Isolation of plant polysomal mRNA by differential centrifugation and ribosome immunopurification methods. Methods Mol Biol 553: 109-126.
  8. Merchante, C., Brumos, J., Yun, J., Hu, Q., Spencer, K. R., Enriquez, P., Binder, B. M., Heber, S., Stepanova, A. N. and Alonso, J. M. (2015). Gene-specific translation regulation mediated by the hormone-signaling molecule EIN2. Cell 163(3): 684-697.
  9. Missra, A. and von Arnim, A. G. (2014). Analysis of mRNA translation states in Arabidopsis over the diurnal cycle by polysome microarray. Methods Mol Biol 1158: 157-174.
  10. Reynoso, M. A., Juntawong, P., Lancia, M., Blanco, F. A., Bailey-Serres, J. and Zanetti, M. E. (2015). Translating Ribosome Affinity Purification (TRAP) followed by RNA sequencing technology (TRAP-SEQ) for quantitative assessment of plant translatomes. Methods Mol Biol 1284: 185-207.
  11. Reuber, T. L. and Ausubel, F. M. (1996). Isolation of Arabidopsis genes that differentiate between resistance responses mediated by the RPS2 and RPM1 disease resistance genes. Plant Cell 8(2): 241-249.
  12. Robinson, M. D. and Oshlack, A. (2010). A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol 11(3): R25.
  13. Robinson, M., McCarthy, D., and Smyth, G. (2010). edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26(1):139-40.
  14. Strimmer, K. (2008). A unified approach to false discovery rate estimation. BMC Bioinformatics 9: 303.
  15. Zanetti, M. E., Chang, I. F., Gong, F., Galbraith, D. W. and Bailey-Serres, J. (2005). Immunopurification of polyribosomal complexes of Arabidopsis for global analysis of gene expression. Plant Physiol 138(2): 624-635.

材料和试剂

  1. 材料
    1. Miracloth
    2. 将30ml Nalgene TM高速聚碳酸酯离心管(Thermo Fisher Scientific,Thermo Scientific ,目录号:3118-0030)(或等价物)离心该植物提取物br />
    3. 5ml聚丙烯薄壁超速离心管(Beckman Coulter,目录号:326819)
    4. 10ml超速离心机,带有等密封盖的14×89mm管(BioComp Instruments,目录号:105-914A)
    5. 细规格
    6. 10毫升注射器
    7. 要连接到注射器上的插管(Thomas Scientific,目录号:1193G13)
    8. 预灭菌,无RNase的5ml,1ml,200μl和10μl微量吸头
    9. 预灭菌,无RNA酶的2ml和1.5ml微量离心管
    10. 15和50ml Falcon管
    11. 塞子固定1.5毫升管的盖子
    12. 剃刀刀片
    13. Dynabeads mRNA纯化试剂盒(Thermo Fisher Scientific,Ambion TM ,目录号:610-06)
    14. 2 ml微量离心管
    15. 1.5ml不粘的,无RNA酶的微管(Thermo Fisher Scientific,Applied Biosystems ,目录号:AM12450)
    16. Myone TM链霉亲和素C1 DynaBeads (Thermo Fisher Scientific,Invitrogen TM ,目录号:65601)
    17. 200μlPCR条带管

  2. 试剂
    1. 液氮
    2. 无RNase,无菌,MilliQ水
    3. Tris碱
    4. 蔗糖
    5. 氯化钾(KCl)
    6. 氯化钠(NaCl)
    7. 氯化镁(MgCl 2)
    8. 乙二醇 - 双(2-氨基乙醚)-N,N,N',N-四乙酸(EGTA)
    9. 乙二胺四乙酸(EDTA)
    10. 二硫苏糖醇(DTT)
    11. Brij-35 [聚氧乙烯(23)月桂基醚]
    12. Triton X-100
    13. Igepal CA 630(辛基苯基 - 聚乙二醇)
    14. 吐温20(聚氧乙烯失水山梨醇单月桂酸酯20)
    15. 环己酰亚胺(见注1)
    16. 氯霉素(见注1)
    17. 氯化锂(LiCl)
    18. 乙酸钠(NaOAc)
    19. 碳酸氢钠(NaHCO 3)
    20. 碳酸钠(Na 2 CO 3)
    21. 十二烷基硫酸钠(SDS)
    22. 氢氧化钠(NaOH)
    23. 柠檬酸三钠(Na 3+,6+,H 5 O,7)
    24. 二甲基亚砜(DMSO),PCR级
    25. 乙醇,分子生物学等级
    26. 水饱和,酸性苯酚,分子生物学等级(见注2)
    27. 氯仿,分子生物学级(见注2)
    28. 异戊醇
    29. 异丙醇,分子生物学等级
    30. PEG8000
    31. 15mg/ml GlycoBlue (Thermo Fisher Scientific,Invitrogen TM ,目录号:AM9515)
    32. 10 bp DNA ladder(Thermo Fisher Scientific,Invitrogen TM ,目录号:10821015)
    33. 2x染色的变性样品缓冲液(Thermo Fisher Scientific,Invitrogen TM ,目录号:LC6876)(参见注释3)
    34. SYBR Gold,在DMSO中10,000x(Thermo Fisher Scientific,Invitrogen TM ,目录号:S11494)(参见注释4)
    35. dNTP混合物,10mM(Thermo Fisher Scientific,Invitrogen TM,目录号:18427-013)
    36. OmniPur聚乙二醇8000(EMD Millipore,Calbiochem ,目录号:6510)
    37. 通用miRNA克隆接头(New England Biolabs,目录号:S1315S)
    38. 硼酸
    39. 乙酸
    40. 12孔15%聚丙烯酰胺TBE-脲凝胶(Bio-Rad Laboratories,目录号:4566055)(参见注释5)
    41. 30%丙烯酰胺/双丙烯酰胺(29:1)(Bio-Rad Laboratories,目录号:161-0156)(见注5)
    42. 过硫酸铵(APS)
    43. TEMED(N,N,N',N'-四甲基乙烷-1,2-二胺)(Sigma-Aldrich,目录号:T7024)
    44. 甘油
    45. 溴酚蓝


    1. SUPERase-In RNase抑制剂(Thermo Fisher Scientific,Ambion TM ,目录号:AM2694)
    2. TURBO TM DNA酶(2U /μl)(Thermo Fisher Scientific,Ambion TM ,目录号:AM2238)
    3. RNA酶I(100U /μl)(Thermo Fisher Scientific,Invitrogen TM ,目录号:AM2294)
    4. SuperScript III逆转录酶(Thermo Fisher Scientific,Invitrogen TM ,目录号:18080-093)
    5. T4多核苷酸激酶,T4 PNK4(New England Biolabs,目录号:M0201),提供有10×T4 PNK缓冲液(New England Biolabs,目录号:B0201)(参见注释6)
    6. T4 RNA连接酶2,截短(New England Biolabs,目录号:M0242),其提供有PEG 8000 50%和10×T4 Rn12缓冲液
    7. Phusion高保真DNA聚合酶(2U /μl)(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:F-530S)
    8. CircLigase TM sup/ssDNA连接酶(100U /μl)(Epicentre,目录号:CL4115K)

  3. 寡核苷酸
    1. 用于分离衔接子环化的逆转录引物(参见注释7)
      NI-NI-9:[P] AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGC [SC18] CACTCA [SC18] TTCAGACGTGTGCTCTTCCGATCTATTGATGGTGCCTACAG(Ingolia等人,2009)
    2. 尺寸标记寡核苷酸(Ingolia等人,2009)(参见注释8)
      oNTI265:rArUrGrUrArCrArCrGrGrArGrUrCrGrArGrCrUrCrArArCrCrCrGrCrArArCrGrCrGrA
      oNTI268:rArUrGrUrArCrArCrGrGrArGrArCrCrCrGrCrArArCrGrCrGrA
    3. rRNA减除寡核苷酸(这里所有的寡核苷酸都具有5'TEG连接的生物素,并且从IDT获得)。这些寡核苷酸基于使用3日龄拟南芥发现的最丰富的rRNA序列和转座子, em>幼苗和本文所述的核糖体印迹协议(参见注释9) rRNABio1:5'-gataaccgtagtaattctagag-3'
      rRNABio2& 4:5'-TGATTCATGATAACTCGACGGACGACGCGGATTACGGTGGCGGC-3'
      rRNABio5& 3:5'-GTCGCTGCCGTGATCGTGGTCTCCATCGAGTCTTTGAACGCAAG-3'
      生物展示:5'-GAGGGATGCAACACGAGGAGTTCCCGGGAGGTCA-3'
      Bio-5S:5'-AAGCCTTCTGGCCGAGGGCACGTCTGCCTGGGTGTCACAA-3'
      Bio-18S:5'-AAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTG-3'
    4. 扩增引物
      NI-NI-2:5'-AATGATACGGCGACCACCGAGATCTACAC-3'(Ingolia等人,2009)
      NI-NI-3:5'-CAAGCAGAAGACGGCATACGAGATAGTCGTGTGACTGGAGTTCAGACGTGTGCTCTTCCG-3'(Ingolia et al。,2009)
      备选索引引物
      索引1:
      5'-CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCG-3'
      索引2:
      5'-CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCAGACGTGTGCTCTTCCG-3'
      索引3:
      5'-CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGACGTGTGCTCTTCCG-3'
      索引4:
      5'-CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTCAGACGTGTGCTCTTCCG-3'
      索引5:
      5'-CAAGCAGAAGACGGCATACGAGATCACTGTGTGACTGGAGTTCAGACGTGTGCTCTTCCG-3'
      索引6:
      5'-CAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAGTTCAGACGTGTGCTCTTCCG-3'
      索引7:
      5'-CAAGCAGAAGACGGCATACGAGATGATCTGGTGACTGGAGTTCAGACGTGTGCTCTTCCG-3'

  4. 解决方案
    所有溶液应使用无RNase,MilliQ水制备。除非另有说明,所有这些产品必须通过高压灭菌至少20分钟,并在室温下储存
    1. 1 M Tris-HCl,pH 7.0(见注10)
    2. 1 M Tris-HCl,pH 8.0(见注10)
    3. 1 M Tris-HCl,pH 9.0(见注10)
    4. 1M蔗糖,由分子生物学级蔗糖制备(高压灭菌10分钟)
    5. 1 M KCl
    6. 5 M NaCl
    7. 1 M MgCl 2
    8. 0.5 M EGTA,pH 8.0(参见注释11)
    9. 0.5 M EDTA,pH 8.0(见注释11)
    10. 2mM EDTA,100mM Na 2 CO 3 Sub Sub
    11. 2mM EDTA,100mM NaHCO 3/v/v
    12. 3 M NaOAc,pH 5.5
    13. 10%SDS(w/v)
    14. 1M NaOH(无需灭菌)
    15. 4 M LiCl(过滤灭菌,不高压灭菌)
    16. 1 M DTT(不要高压灭菌;等分并储存在-20℃)
    17. 100mg/ml环己酰亚胺,在DMSO中制备(过滤灭菌;等分并储存在-20℃)
    18. 50mg/ml氯霉素,在乙醇中制备(过滤灭菌;等分并储存在-20℃)
    19. 洗涤剂混合物中的四种洗涤剂(Brij-35,Triton X-100,Igepal CA 630和Tween 20)中的每一种的20%(w/v或v /
    20. 50%PEG8000(w/v),通过将干PEG与水在50ml锥形中混合并轻轻振荡混合过夜制备。不需要灭菌。 PEG非常吸湿;因此开始添加PEG至少于MilliQ水的最终体积的一半。然后,溶解后,如果需要,加入更多的水。用于结扎的PEG(步骤C3)不应超过1个月大
    21. 10%APS(w/v),过滤灭菌并小份等分(例如,500μl)。储存于-20°C。等分试样可以解冻并重新冷冻几次。

  5. 缓冲区(参见配方)
    1. 多聚体提取缓冲液(PEB)
    2. 蔗糖垫A(SCA)
    3. 蔗糖梯度解决方案
    4. 多聚体消化缓冲液(PDB)
    5. 蔗糖垫B(SCB)
    6. Polysome重悬浮缓冲液(PRB)
    7. 总RNA提取缓冲液(TREB)
    8. 碱性片段缓冲液(2x)
    9. 碱性碎裂停止/沉淀溶液
    10. TAE(Tris /乙酸盐/EDTA缓冲液)(50x)
    11. TBE(Tris /硼酸盐/EDTA缓冲液)缓冲液(5x)
    12. RNA凝胶提取缓冲液(GEB)
    13. DNA凝胶提取缓冲液(STE)
    14. SSC(20x)
    15. 减法结合/洗涤缓冲液(2x)
    16. 8%的非变性聚丙烯酰胺凝胶(12ml)
    17. 非变性负载染料(6x)

设备

  1. 7-9厘米直径的陶瓷砂浆和杵
  2. 小(30-50 ml)玻璃烧杯
  3. 冷冻Beckman Avanty J-25离心机和Beckman JA17转子(或同等品)
  4. 使用具有用于多核糖体制粒(Beckman Coulter,型号:SW55Ti)和蔗糖梯度离心(Beckman Coulter,型号:SW41Ti或Thermo Fisher Scientific,Thermo Scientific)的摇摆桶转子的超速离心机Beckman L8-70M(或等同物) ,型号:TH-641)
  5. 自动P1000,P200和P10微量移液器
  6. 制冷台式微量离心机
  7. 轨道振动器
  8. 梯度主站,使用梯度制备器和分馏站(BioComp Instruments)
  9. 连续UV光监测器(Bio-Rad Laboratories,型号:EM-1 Econo UV监测器)
  10. 分馏器(Gilson,型号:FC203B)
  11. Nanodrop(或另一个分光光度计,用于定量小体积RNA中的核酸浓度)
  12. 营养振动器
  13. 热块
  14. 通风橱
  15. Mini-PROTEAN四细胞聚丙烯酰胺凝胶盒(Bio-Rad Laboratories,目录号:165-8004)或等同物和电泳电源
  16. 紫外透射仪
  17. 涡流
  18. DynaMag TM -2磁性分离架(Thermo Fisher Scientific,目录号:12321D)
  19. 温度计
  20. 2100 BioAnalyzer(Agilent Technologies,目录号:G2940CA)
  21. HiSeq2000(Illumina)

程序

湿实验室方案分为A,B和C步骤(参见图1的概述)。关于制备核糖体印迹的步骤。 B步骤是指总mRNA分离程序。 C步骤代表方案的部分,其中将足迹和mRNA片段一起加工以产生测序文库。执行A和B步骤的顺序取决于研究者。
组织需要在所需的处理后快速冷冻,在液氮中研磨成非常细的粉末,然后在-80℃下储存直至使用。 0.5μg的这种粉碎的组织是核糖体印迹所需的,并且需要0.5-1g来制备mRNA片段。优良作法是首先分离多核糖体而不包括消化步骤以确保在进行覆盖库的制备之前分离完整的多核糖体。与所有RNA协议一样,在任何时候不应该允许组织在没有缓冲液的情况下解冻,并且所有材料和溶液需要不含RNA酶。除非另有说明,所有的工作都应在冰上进行。
这些协议允许在9天内从冷冻的地面组织产生准备好的Ribo-seq和RNA-seq文库。整个协议是在工作日组织的。在方案结束时,我们包括一个数据分析部分,其描述了通过治疗或突变而导致翻译水平调节的基因的鉴定的管道。


图1.核糖体足迹程序概述。A.蓝色面板:核糖体足迹特异性步骤; B.绿色面板:特异于mRNA文库制备的步骤; C.黄色面板:两种类型的库都通用的步骤。 A,B和C步骤也在书面协议中标记。

  1. 步骤:核糖体足迹
    图2示出了包括A步骤的过程的流程图

    图2.协议的A步骤概述

-top:0px; margin-bottom:0px;> < style ="margin-bottom:0px;>

数据分析

本文描述的使用核糖体印迹数据研究翻译调节的生物信息学分析遵循Merchante等人描述的管道。 (2015)。该管道的输入是来自Illumina HiSeq2000平台的解复用Ribo-seq和RNA-seq数据集(fastq文件),TAIR 10拟南芥基因组序列和TAIR 10拟南芥基因模型( https://www.arabidopsis.org/)。

  1. 一般数据预处理
    1. 使用FASTX工具包( http://hannonlab.cshl.edu/fastx_toolkit/),删除衔接头序列和修剪低质量区域
    2. 使用Tophat2( https://ccb.jhu.edu/software/tophat/index.shtml )。
    3. 选择唯一映射,大小范围为20-40 nt的感应方向读取,丢弃具有两个以上不匹配的对齐,并创建编码(CDS)区域的读取计数摘要。

  2. 质量控制周期的评估
    核糖体足迹,但不是RNA-seq读数,应该 显示强的3nt周期性,其代表密码子长的逐步 核糖体沿mRNA的运动(Ingolia等人,2009; Merchante等人, 2015)。所以,这种周期性的存在可以用作控制 质量的足迹。为了避免边缘效应,限制分析 具有长于70nt的5'和3'UTR的基因和长于150nt的CDS。至 避免关于沿着转录物的读取位置的模糊性, 将分析限制为固定长度的读取 - 通常为28至之间的值 32 nt,通过视觉检查(C1步骤)确定 - 在意义上的映射 方向只有一个抄本。
    1. 将基因组阅读坐标转换为转录本坐标
    2. 可视化CDS开始:项目附近的读取分布选择的读取到"metagene"的公共坐标系。使用它们相对于主开放阅读框的开始的相对位置来对读取起始点的转录物坐标进行二进制化:在CDS的开始处开始的读取落入bin'0'。为了可视化CDS末端周围的读取分布,以类似的方式进行,但是相对于主开放阅读框的末尾处理。
    3. 通过所选读取的总数来标准化读取开始位置的直方图,并绘制一个围绕bin'0'的区域
  3. 差异性转录和翻译的边缘分析
    1. 丢弃具有小读数的基因(任何样品中rpkm值小于3),并执行TMM标准化(Robinson和Oshlack,2010)。
    2. 通过2-因子设计建模实验,其中因子1是例如乙烯/对照处理,因子2是RNA-seq/Ribo-seq处理。使用Bioconductor package edgeR(Robinson等人,2010)的广义线性模型方法(glm edgeR)选择在该实例中i)在RNA水平上响应乙烯的基因,ii )在足迹水平上响应乙烯,以及iii)在RNA水平和足迹水平上对乙烯的响应不同。使用Benjamini和Hochberg的方法(Benjamini和Hochberg,1995)确定假发现率。
     
  4. 分析变化 翻译效率
    1. 根据Ingolia及其同事的定义(Ingolia等人,2009),将计算翻译效率(TE)作为Ribo-seq足迹rpkm值相对于RNA-seq rpkm值的分数:TE = rpkm脚)/rpkm(mRNA)
    2. 计算乙烯处理的植物的log 2变换的TE值,并减去相应的对照值:ΔTE= log 2(TE亚乙基)-log 2 (TE control
    3. 计算ΔTE的z分数:zΔTE=(ΔTE-E [ΔTE])/√Var(ΔTE)。使用R包fdrtool确定错误发现率(Strimmer,2008)。

笔记

  1. 环己酰亚胺和氯霉素毒性很大,对环境有害
  2. 苯酚和氯仿均为急性毒素。在通风橱中对其进行处理,并采取适当的保护措施。
  3. 具有染料的2x变性样品缓冲液含有甲酰胺,其是生殖毒素。在处理时应采取适当的保护措施。
  4. 核酸污渍通常是致突变的,在处理它们时使用适当的保护
  5. 丙烯酰胺和双丙烯酰胺都是神经毒素,在处理它们时使用适当的保护
  6. 确保不要与T4多核苷酸激酶3'磷酸酶减(M0236)混淆正确的T4 PNK(M0201),后一种情况下,连接步骤将不起作用。
  7. [P]表示5'-磷酸化,[SC18]表示六乙二醇间隔基
  8. 'r'表示这些是RNA寡核苷酸
  9. 如果使用不同的处理,生长条件,发育阶段,足迹协议等,这些寡核苷酸可能需要重新设计。
  10. 该方案需要在pH7.5,7.8,8.0,8.4,8.8和9.0的Tris-HCl,但是所有中间浓度的Tris-HCl缓冲液可以从这里列出的Tris-HCl pH7.0,8.0和9.0制备。
  11. EGTA和EDTA仅在调节pH后溶解
  12. 由于使用洗涤剂,会有泡沫。确保收集所有的气泡。
  13. 当制备等分试样时,允许一些过量,因为溶液非常粘稠。对于两个样品,1.2ml等分试样工作良好
  14. SW55Ti转子允许同时旋转6个样品,因此每次离心一轮最多需要3ml SCA。
  15. 例如5ml超速离心管或15ml Falcon管
  16. 该步骤是必要的,因为如果上清液在旋转结束时没有快速转移到不同的管中,则碎屑小球可以容易地脱落;未能立即转移上清液将导致碎屑的携带并损害超速离心
  17. 蔗糖梯度也可以在单体造粒离心机 - 步骤A2.7制备
  18. 大约一半的管。
  19. 如果使用光合组织,多核糖体丸将是清澈的,如果使用光合组织,则是深绿色
  20. 如果有一个控制管不会被消化,不要加RNase I到它,并保持在冰上,直到消化时间结束。
  21. 执行此操作时请格外小心,以免干扰渐变。
  22. 如果使用1.5 ml管加热苯酚:氯仿,不要让温度高于65°C并固定盖子,因为由于压力,盖子可以很容易打开并产生危险的溢出,导致损失了珍贵的样品
  23. 单独的苯酚也可以用于提取RNA,但是如果是这种情况,水相可以是较低的,因为提取物中高浓度的蔗糖。
  24. 也可以使用与高通量测序文库构建和下一代测序兼容的其他标准RNA提取方法(例如,Trizol)。
  25. 苯酚:氯仿:异戊醇(25:24:1)也可以使用
  26. 也可以使用Beckman Avanty J-25中的JA17管
  27. 如果使用2ml离心管,加入500μl提取物到不同的微管预先填充50μl3 M乙酸钠pH 5.2,混合和添加1375微升乙醇。
  28. 34,895 x g 如果使用Beckman离心机
  29. 在此步骤中,如果需要,样品可以在4℃下放置过夜
  30. 停止溶液已含有300mM盐,因此不需要更多的盐来沉淀RNA
  31. 如果样品被寡核苷酸包围,则更容易选择大小,因此,在凝胶的开始和结束处运行两个寡聚体泳道。
  32. 运行的时间必须根据运行的单元根据经验确定。使用引物开始运行测试是一个好的做法。作为参考,对于第一次运行不让来自变性样品缓冲液的下蓝色带跑出凝胶。注意标记引物相对于染料的位置,并调整未来运行时间以进行引物测试
  33. 凝胶块不破碎或改变外观。
  34. 允许相对较高的过量,因为混合物非常粘稠,难以移液
  35. 如果室温孵育后在4℃过夜孵育,连接效率增加
  36. 作为参考,连接的样品通常在样品缓冲液的较高蓝色带上方运行
  37. 当引物以大量过量存在时,来自未延伸引物的背景增加,其在一些情况下可支配实际文库产物。最好根据经验确定正确的比例。 1-2μl1.25μM的NI-NI-9引物应该正常工作。这可以在凝胶运行后测试:未延伸的引物在RT步骤后仍然存在,但是扩展文库不应比引物本身更暗。
  38. 将环化的文库重悬于20μl中,仅10μl用于减去杂交步骤。将剩余的10μl保持在-80°C作为备用
  39. 减法杂交中的缓冲条件(高盐以及螯合镁的柠檬酸盐)与PCR不相容,因此在减法步骤后需要沉淀。
  40. 寻找导致最佳扩增而不显着损失引物的循环数,使得引物的序列之间不存在竞争。条件在实验之间通常是稳定的。因此,一旦已经为样品子集确定了最佳条件,它们也证明适合于来自不同实验的其他样品。在我们的情况下,在14个循环时达到最佳扩增,并且使用另外的样品的进一步测试确认了14个循环为最佳,而不需要进一步优化。
  41. 保持PCR的其余部分作为备份

食谱

  1. 多聚体提取缓冲液(PEB)
    110 mM Tris-HCl,pH 8.8
    100mM蔗糖 100 mM KCl
    75mM NaCl 20mM MgCl 2/
    12.5 mM EGTA
    3 mM DTT
    6.25μl/ml洗涤剂混合物
    25μl/ml Triton X-100 37.5μg/ml放线菌酮 25μg/ml氯霉素
    提取前准备新鲜,并保持冰上,直到使用
  2. 蔗糖垫A(SCA)
    1.75 M蔗糖
    400mM Tris-HCl,pH9.0
    200 mM KCl 
    5mM EGTA
    35mM MgCl 2
    5 mM DTT
    50μg/ml放线菌酮 50μg/ml氯霉素
    提前准备(减去DTT和抗生素)。高压灭菌不超过15分钟,使1.2毫升等分,并存储在-20°C。在使用前添加每个实验新鲜的最后三种化合物。
  3. 蔗糖梯度解决方案
    50%/10%(w/v)蔗糖 40mM Tris-HCl,pH 8.4
    20 mM KCl
    10mM MgCl 2/
    50μg/ml放线菌酮 50μg/ml氯霉素
    1μg/ml SUPERase-In
    在实验当天准备新鲜的
  4. 多聚体消化缓冲液(PDB)
    20mM Tris-HCl,pH 7.8
    150mM NaCl 5mM MgCl 2/
    50μg/ml Triton X-100 1 mM DTT
    100μg/ml放线菌酮
    12μlTURBO DNase 2 U /μl/4.5 ml消化体积
    10μlRNase I/4.5ml消化缓冲液
    准备在提取的日子,并保持冰
  5. 蔗糖垫B(SCB)
    1.75 M蔗糖
    20mM Tris-HCl,pH 7.8
    150mM NaCl 5mM MgCl 2/
    5 mM DTT
    50μg/ml放线菌酮 50μg/ml氯霉素
    1μl/ml SUPERase-In
    提前准备(减去最后四种成分),高压灭菌不超过15分钟。使1.2毫升等分,存储在-20°C。在使用前,为每个实验新添加最后四种化合物。
  6. Polysome重悬浮缓冲液(PRB)
    200mM Tris-HCl,pH 8.4
    200 mM KCl
    25mM EGTA
    35mM MgCl 2
    50μg/ml放线菌酮 50μg/ml氯霉素
    1μl/ml SUPERase-In
    准备提取的日子,并保持在冰
  7. 总RNA提取缓冲液(TREB)
    200mM Tris-HCl,pH8.0 400mM LiCl 25mM EDTA
    1%SDS
    提前准备(溶液保存几个月),并在室温下保存
  8. 碱性片段缓冲液(2x)
    2mM EDTA 100mM Na 2 CO 3,pH 9.2 该溶液通过将15份100mM Na 2 CO 3和110份100mM NaHCO 3混合来制备。它将与气态CO 2平衡并随时间升高pH。它可以在密封的一次性使用的等分试样在室温下储存一年以上
  9. 碱性碎裂停止/沉淀溶液
    60微升3 M NaOAc
    2μlGlycoBlue 15 mg/ml
    每次反应500μl无RNA酶的水
    在使用前准备
  10. TAE(Tris /乙酸盐/EDTA缓冲液)(50x) 2 M Tris碱
    1M乙酸
    50 mM EDTA
  11. TBE(Tris /硼酸盐/EDTA缓冲液)(5x)
    445mM Tris碱
    445mM硼酸 10 mM EDTA
    使用前将浓缩的储备缓冲液稀释至1倍。
  12. RNA凝胶提取缓冲液(GEB)
    300mM NaOAc,pH 5.5 1mM EDTA
    0.25%SDS
  13. DNA凝胶提取缓冲液(STE)
    300 mM NaCl
    10mM Tris-HCl,pH8.0 1mM EDTA
  14. SSC(20x)
    3 M NaCl
    300mM柠檬酸三钠(Na 3 H 6 H 5 O 7)。
    用HCl调节至pH7.0
  15. 减法结合/洗涤缓冲液(2x)
    400μl5M NaCl
    2μl0.5M EDTA
    10μl1M Tris-HCl,pH 7.5
    590μl水
  16. 8%的非变性聚丙烯酰胺凝胶(12ml) 6.3ml MilliQ水
    3.2ml 30%丙烯酰胺/双丙烯酰胺 2.4 ml 5x TBE
    84微升10%APS
    10μlTEMED
  17. 非变性负载染料(6x)
    60%(v/v)甘油 0.03%(w/v)溴酚蓝

致谢

这项工作是支持的NSF拨款MCB 1158181和0519869到J.M.A。 MCB 0923727 to J.M.A.和A.N.S。 IOS 1444561 to J.M.A.,A.N.S.,and S.H。 NCSU-RISF到S.H.和J.M.A。和玛丽·居里COFUND U-移动博士后研究金C.M. (由马拉加大学和欧盟7FP GA N_246550共同资助)。我们感谢Nick Ingolia对在工厂中实施和解决这个协议的帮助。

参考文献

  1. Branco-Price,C.,Kaiser,KA,Jang,CJ,Larive,CK和Bailey-Serres,J.(2008)。  控制错误发现率:一种实用且强大的多重测试方法。 R. Statist。 Soc。 B 57:125-133
  2. Ingolia,NT,Ghaemmaghami,S.,Newman,JR和Weissman,JS(2009)。  基因组范围注释和通过核糖体分析进行的翻译定量。 Curr Protoc Mol Biol Chapter 4:Unit 4 18.  br />
  3. Juntawong,P.,Girke,T.,Bazin,J.和Bailey-Serres,J.(2014)。  通过拟南芥中核糖体足迹的全基因组分析揭示的转化动力学。


    Proc Natl Acad Sci USA 111(1):E203-212。
  4. Li,R.,Sun,R.,Hicks,GR和Raikhel,NV(2015)。  拟南芥核糖体蛋白通过调节脂质代谢来控制液泡运输和发育程序。 Proc Natl Acad Sci USA 1):E89-98。
  5. Mustaping,A.,Juntawong,P.和Bailey-Serres,J.(2009)。  通过差速离心和核糖体免疫纯化方法分离植物多核糖体mRNA。 Methods Mol Biol 553:109-126。
  6. Merchante,C.,Brumos,J.,Yun,J.,Hu,Q.,Spencer,KR,Enriquez,P.,Binder,BM,Heber,S.,Stepanova,ANand Alonso,JM ; 由激素信号分子EIN2介导的基因特异性翻译调节。 单元格 163(3):684-697。
  7. Missra,A.和von Arnim,A.G。(2014)。 拟南芥中的mRNA翻译状态分析通过多核糖体微阵列进行的昼夜周期。 1158:157-174。
  8. Reynoso,MA,Juntawong,P.,Lancia,M.,Blanco,FA,Bailey-Serres,J.and Zanetti,ME(2015)。  翻译核糖体亲和纯化(TRAP),随后是RNA测序技术(TRAP-SEQ),用于植物翻译平台的定量评估。 > Methods Mol Biol 1284:185-207。
  9. Reuber,TL和Ausubel,FM(1996)。  隔离的拟南芥基因,其区分由RPS2和RPM1疾病抗性基因介导的抗性反应。植物细胞 8(2):241-249。 >
  10. Robinson,MD和Oshlack,A。(2010)。  用于RNA-seq数据的差异表达分析的定标归一化方法。基因组生物学11(3):R25。
  11. Robinson,M.,McCarthy,D。和Smyth,G。(2010)。  edgeR:用于数字基因表达数据的差异表达分析的Bioconductor包。生物信息学26(1):139-40。
  12. Strimmer,K.(2008)。  统一的方法假发现率估计。
  13. Zanetti,ME,Chang,IF,Gong,F.,Galbraith,DW和Bailey-Serres,J.(2005)。  拟南芥的多核糖体复合物的免疫纯化用于基因表达的全局分析植物生理学 138 2):624-635。
English
中文翻译

免责声明

为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。

X


How to cite this protocol: Merchante, C., Hu, Q., Heber, S., Alonso, J. and Stepanova, A. N. (2016). A Ribosome Footprinting Protocol for Plants. Bio-protocol 6(21): e1985. DOI: 10.21769/BioProtoc.1985; Full Text



可重复性反馈:

  • 添加图片
  • 添加视频

我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

问题&解答:

  • 添加图片
  • 添加视频

(提问前,请先登陆)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


登陆 | 注册
引用格式
分享
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook