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Preparation of Multiplexed Small RNA Libraries From Plants
制备植物复用小型RNA库   

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Abstract

High-throughput sequencing is a powerful tool for exploring small RNA populations in plants. The ever-increasing output from an Illumina Sequencing System allows for multiplexing multiple samples while still obtaining sufficient data for small RNA discovery and characterization. Here we describe a protocol for generating multiplexed small RNA libraries for sequencing up to 12 samples in one lane of an Illumina HiSeq System single-end, 50 base pair run. RNA ligases are used to add the 3’ and 5’ adaptors to purified small RNAs; ligation products that lack a small RNA molecule (adaptor-adaptor products) are intentionally depleted. After cDNA synthesis, a linear PCR step amplifies the DNA fragments. The 3’ PCR primers used here include unique 6-nucleotide sequences to allow for multiplexing up to 12 samples.

Keywords: Small RNA(小核糖核酸), High-throughput sequencing(高通量测序技术), Library preparation(文库制备), Multiplexing(复用), SRNA-seq(sRNA序列)

Materials and Reagents

  1. Low molecular weight gel-based small RNA isolation
    1. ssRNA marker (Takara Bio Company, catalog number: 3416 )
    2. Reagents for 17% polyacrylamide, 7M Urea PAGE
      1. 37.5:1 polyacrylamide:bisacrylamide (see Recipes)
      2. Urea (Life Technologies, catalog number: 15505-050 )
      3. TEMED (Life Technologies, catalog number: 15524-010 )
      4. 10% ammonium persulfate (Sigma-Aldrich, catalog number: A3678-100G ) (see Recipes)
    3. 2x RNA loading dye (see Recipes)

  2. DE81-based nucleic acid purification
    1. Fiber glass (Corning Incorporated, catalog number: 988-10144 )
    2. Extra thick blot paper (Bio-Rad Laboratories, catalog number: 170-3969 )
    3. Whatman DE81 ion exchange cellulose chromatography paper (Thermo Fisher Scientific, catalog number: 05-717-1A )
    4. GlycoBlue coprecipitant (Life Technologies, catalog number: AM9516 )
    5. 100% ethanol
    6. 75% ethanol
    7. EB buffer (QIAGEN, catalog number: 19086 )
    8. Qubit RNA HS Assay Kit for quantification of purified small RNAs (Life Technologies, catalog number: Q32852 )
    9. Qubit dsDNA HS Assay Kit for quantification of DNA amplicon (Life Technologies, catalog number: Q32854 )
    10. Low salt buffer (see Recipes)
    11. High salt buffer (see Recipes)

  3. Multiplexed small RNA libraries preparation
    1. 8-Strip PCR thin-walled, 200 µl tubes (Corning Incorporated, Axygen®, catalog number: PCR-0208-CP-C )
    2. miRNA cloning linker 1 (/5'App/CTGTAGGCACCATCAAT/3'ddC/; 1 nm) (Integrated DNA Technologies, catalog number: 11-04-03-05 )
    3. Truncated, K227Q mutation T4 RNA ligase 2 (New England Biolabs, catalog number: M0351L )
    4. De-adenylase (New England Biolabs, catalog number: M0331S )
    5. Exonuclease VII (United State Biological, catalog number: 70082Z )
    6. RNA 5’ adapter (GUUCAGAGUUCUACAGUCCGACGAUC) (Illumina RA5, catalog number: 15013205 )
    7. dATP (Life Technologies, catalog number: 55082 )
    8. T4 RNA ligase I (Life Technologies, catalog number: AM2141 )
    9. RT-PCR primer (ATTGATGGTGCCTACAG; 25 nmol; de-salted) (Integrated DNA Technologies)
    10. SuperScript III (Life Technologies, catalog number: 18080051 )
    11. Phusion high fidelity II (Thermo Fisher Scientific, catalog number: F549L )
    12. 5' PCR primer (Illumina small RNA PCR primer 2: AATGATACGGCGACCACCGACAGGTTCAGA-GTTCTACAGTCCGA)
    13. 3' indexed PCR primer I1 - I12 (100 nmol; PAGE-purified) (Integrated DNA Technologies)
      Note: The barcodes below (underlined sequences) are reverse complemented in the final Illumina output sequence (see sequence in square brackets for each primer).
      I1 [CGATGT]: CAAGCAGAAGACGGCATACGAACATCGATTGATGGTGCCTACAG
      I2 [GATCAC]: CAAGCAGAAGACGGCATACGAGTGATCATTGATGGTGCCTACAG
      I3 [CAGATG]: CAAGCAGAAGACGGCATACGACATCTGATTGATGGTGCCTACAG
      I4 [TACGTT]: CAAGCAGAAGACGGCATACGAAACGTAATTGATGGTGCCTACAG
      I5 [TTACCA]: CAAGCAGAAGACGGCATACGATGGTAAATTGATGGTGCCTACAG
      I6 [ACTGTA]: CAAGCAGAAGACGGCATACGATACAGTATTGATGGTGCCTACAG
      I7 [ATCACG]:  CAAGCAGAAGACGGCATACGACGTGATATTGATGGTGCCTACAG
      I8 [ACTTGT]:  CAAGCAGAAGACGGCATACGAACAAGTATTGATGGTGCCTACAG
      I9 [GCCAAT]:  CAAGCAGAAGACGGCATACGAATTGGCATTGATGGTGCCTACAG
      I10 [TGCTAG]:  CAAGCAGAAGACGGCATACGACTAGCAATTGATGGTGCCTACAG
      I11 [CTTGTA]:  CAAGCAGAAGACGGCATACGATACAAGATTGATGGTGCCTACAG
      I12 [TCAGGC]:  CAAGCAGAAGACGGCATACGAGCCTGAATTGATGGTGCCTACAG
    14. QIAquick PCR Purification Kit (QIAGEN, catalog number: 28106)
    15. Diethylpyrocarbonate (DEPC)-H2O/RNase-free H2O
    16. DNA size marker suitable for detecting a ~120 bp fragment (50 bp step ladder, Promega Corporation, catalog number: G4521 ; 25 bp step ladder, Promega Corporation, catalog number: G4511 )
    17. 3 M sodium acetate (NaOAc) (pH 5.5)
    18. Reagents for 6% native PAGE
      1. 37.5:1 polyacrylamide:bisacrylamide (see Recipes)
      2. TEMED (Life Technologies, catalog number: 15524-010)
      3. 10% ammonium persulfate (Sigma-Aldrich, catalog number: A3678-100G) (see Recipes)

Equipment

  1. Thermocycler
  2. Microcentrifuge: For use at room temperature and at 4 °C
  3. Fisher gel-loading tips (Thermo Fisher Scientific, catalog number: 02-707-182 )
  4. Agarose gel electrophoresis systems (Bio-Rad Laboratories, Sub-Cell GT system)
  5. Polyacrylamide gel electrophoresis system (Thermo Fisher Scientific, owl Dual-Gel vertical electrophoresis systems)
  6. 65 °C water bath
  7. Semi-dry blotting system (Sigma-Aldrich, semi-dry blotter, catalog number: Z340502 )
  8. Heating block

Procedure

Part I. Low molecular weight gel-based small RNA isolation
Notes:

  1. This step is not required if you already purified your small RNAs (e.g. by immunoprecipitation). See note A in Notes section. In this case, skip to section C.
  2. Duration of this step:
    1. Gel preparation and polymerization: 1+ h.
    2. Gel pre-run time: 30+ min.
    3. Gel running time: 4+ h, plus loading time and staining.

  1. Preparing a 17% polyacrylamide gel containing 7 M Urea in 0.5x TBE
    1. The day before you plan to run the gel, assemble glass plates and cover with Saran wrap to protect.
      1. Use plates of 14 cm x 16 cm and 1.5 mm spacers.
      2. Clean all materials (plates, spacers and comb).
        1. Wash with 10% sodium dodecyl sulfate (SDS).
        2. Rinse thoroughly with distilled-water.
        3. Rinse with 95% ethanol.
        4. Allow to air-dry.
    2. While preparing polyacrylamide gel mix (below): Seal the outside edges of your plate set-up with 2% agarose (55 °C) to prevent leaking. Sealing is achieved in a few minutes.
    3. Prepare the mix (30 ml) for making one 17% denaturing Urea-PAGE/0.5x TBE gel with 15-well comb by combining the following:
      Reagent (stock concentration)
      Volume (final concentration)
      Acrylamide:bis 37.5:1 solution (30%)
      17 ml (17%)
      Urea
      12.6 g (7 M)
      TBE (10x)
      1.5 ml (1x)
      H2O
      2.0 ml
    4. Mix the above together thoroughly by inversion.
    5. Heat to 65 °C for 10 min to dissolve the urea; invert to mix every ~2 min to help the process.
    6. Let stand on bench for ~10 min then transfer to an ice bath in the cold room (4 °C) for 15+ min.
    7. Add the remaining reagents:
      Reagent (stock concentration)
      Volume (final concentration)
      TEMED
      25 µl *Mix thoroughly by inversion
      APS (10%)
      150 µl (0.05%) *Mix thoroughly by inversion
    8. Pour the gel and allow it to polymerize for at least 30 min; should see beginning of polymerization within ~4 min.
    9. Assemble your electrophoresis device and add the running buffer (0.5x TBE) to inside and outside chambers.
    10. Rinse out wells with a syringe; rinse away bubbles along bottom edge of glass plates.
    11. Gel must be pre-run (at 180 V) for 30-60 min to remove all traces of APS (ammonium and persulfate ions), to distribute/equilibrate any stabilizing factors or ions that were added to the running buffer, and to ensure a constant gel temperature during the run of the samples. Occurrence of concave front of bands is reduced when pre-running the gel. If desired, a blank solution can be loaded: Mix equal amounts of DEPC-H2O and 2x loading dye. 15 µl/lane is sufficient.

  2. Preparing RNA samples and running the gel
    *Keep sample volume to a minimum for best resolution (≤50 µl).
    1. Prepare samples, markers, ladders and blanks in equal volume using 2x RNA loading dye at a final concentration of 1x.
    2. Heat samples at 65 °C for 10 min and immediately quench on ice. Spin briefly to collect sample at the bottom of tube.
    3. Load samples.
      Note: Before loading samples clean the lanes with a syringe to remove the excess of urea. Sample loading determines the ultimate quality of your gel. We use Fisher gel-loading tips to load small RNA gels. Load the sample at the very bottom of the well in a flat band, no triangles, blobs, or other sloppy loading.
    4. Run at 180 V in 0.5x TBE buffer until the bromophenol blue (BPB) dye reaches the bottom of the gel (~4 to 4.5 h).
    5. Stain gel with ethidium bromide in 0.5x TBE buffer.
    6. See below for eluting RNA from acrylamide fragment using DE81 paper.

Part II. DE81-based nucleic acid purification
Notes: Duration of this step

  1. Excision of gel slices and semi-dry transfer: 40+ min.
  2. Elution (RNA): 60+ min (day 1); precipitation #1: overnight; precipitation #2: 2.5+ h.
  3. Elution (DNA): 60+ min (day 1); precipitation #1: 1+ h; precipitation #2: 2+ h.

  1. Prepare elution tubes
    1. Remove the cap from a 500 µl microcentrifuge tube and poke a hole in the bottom center of the tube with a 25-gauge needle (Figure 1A). Label the tube.
    2. Stuff ~25-40 mg of sterile glass wool in the bottom (not through the hole) of the tube using a sterile toothpick or pipette tip. Compact the glass wool to just below the 0.1 ml graduation mark of the tube to leave room for the DE81 paper and buffer (Figure 1B).
    3. Remove the cap from a 2 ml microcentrifuge tube (Figure 1C) and place the prepared 500 µl tube (Figure 1D) inside.


      Figure 1. Preparation of the elution tubes. A. A 500 µl microcentrifuge tube has the cap removed and a hole made in the bottom center. B. Glass wool is placed inside the tube and compacted, leaving space for the buffer and DE81 paper. C. A 2 ml microcentrifuge tube has the cap removed. D. The prepared 500 µl tube is placed inside the 2 ml tube and is ready for use.

  2. Nucleic acid excision and transfer
    1. Place one thick Whatman paper, pre-soaked with 0.5x TBE, on semi-dry blotter; roll with a test tube or 10 ml pipet to remove any air bubbles.
    2.  Excise desired fragment from gel (Figure 2) and place on a piece of DE81 paper labeled using a pencil. Each sample/replicate should be placed onto its own DE81 paper. DE81 paper contains diethylaminoethyl (DEAE) cellulose functional groups that will bind nucleic acids, which can be reversed by the addition of a high salt buffer (Dretzen et al., 1981).
      Include a transfer control - i.e. transfer a gel fragment containing bands from the Ladder lane.


      Figure 2. 17% Polyacrylamide-Urea gel for small RNA isolation stained with ethidium bromide. A. Total RNA before excision of small RNAs. B. Same lane, with small RNAs (18 - 26 nt) excised. ssRNA marker sizes are indicated.

    3. Place paper + gel onto thick Whatman paper on blotter.
    4. Place a second piece of pre-soaked thick Whatman paper on top.
    5. Carefully roll the top of the second Whatman paper to remove any air bubbles.
    6. Transfer 20 min at 400 mAmp in semi-dry blotter.
    7. Remove the top Whatman paper.
      1. Verify transfer by examining the gel fragment and DE81 paper of the marker of the transfer control, side by side, on a UV trans-illuminator. Nucleic acid should not be visible in the gel fragment (Figure 3A) but should be visible on the DE81 paper (Figure 3B). Transfer of ssRNA markers can be difficult to visualize.


        Figure 3. Verification of the nucleic acid transfer. A. Gel fragment is outlined and no nucleic acid is visible. B. DE81 paper with visible nucleic acid transferred (50 bp DNA step ladder).

      2. While the gel + paper is still on the bottom Whatman paper, use a razor to trim away the excess DE81 paper. Discard gel slice and carefully stuff the DE81 paper into the elution tube containing glass wool.

  3. Nucleic acid elution
    1. Thoroughly submerge DE81 paper with ~200 µl (usually 200-500 µl) of low salt buffer. Use pipet tip to gently push paper as low as possible so it is completely covered by the buffer.
    2. Spin 30 sec at maximum speed in a tabletop microcentrifuge. Discard flow-through.
    3. Repeat low salt buffer wash twice for a total of three washes.
    4. Transfer upper column/paper assembly to a clean, labeled 2 ml tube with cap removed.
    5. Elution:
      1. Add 150 µl high salt buffer to paper, and submerge it.
      2. Incubate at 70 °C for 15 min - use foil to cover the tubes. Do not use a water bath.
      3. Spin briefly (10 sec) in a tabletop microcentrifuge and save flow through.
    6. Place a new 2 ml tube beneath filter assembly and repeat step C5 (elution) twice more.
    7. Ethanol precipitation:
      1. Combine the three eluates and add 20 µg glycogen (e.g. GlycoBlue).
      2.  Add 2.5 volumes 100% ethanol and mix. Ideally the precipitation volumes fit in a single 1.5 ml microcentrifuge tube. Using 150 µl of high salt buffer + ~1,150 µl ethanol will just fit in a 1.5 ml microcentrifuge tube = more efficient precipitation.
      3. Precipitate at -80 °C: Overnight for RNA samples; 1+ h for DNA samples.
      4. Centrifuge at maximum speed in a refrigerated microcentrifuge:
        1. 35 min for RNA samples.
        2. 15 min for DNA samples.
      5. Pour off ethanol and wash pellet with 75% ethanol.
      6. Spin 5 min at maximum speed in refrigerated microcentrifuge.
      7.  Remove ethanol and drain tube upside-down on a clean KimWipe for 10 min. If necessary, use a clean KimWipe to remove excess liquid from the inside of the tube - avoid touching the (blue) pellet.
      8. Resuspend pellet in 40 µl DEPC-treated water (if multiple pellets for one sample, pool pellets at this step into a final volume of 40 µl).
    8. A second precipitation is required to remove residual salt for downstream enzymatic steps:
      1. Add 4 µl 3 M NaOAc. Add 100 µl 100% ethanol. Mix. Precipitate at -80 °C for at least 2 h (or overnight)
        1. Follow steps C7d-g.
        2. Resuspend final pellet in 10-12 µl of DEPC-H2O for RNA or 10-12 µl of EB buffer for DNA.
    9. If isolating small RNAs: RNA is now ready to be a template for the Multiplexed small RNA libraries preparation step (section C below).
      1. Quantification: 2 µl + Qubit RNA HS Assay Kit. For samples where the Qubit returns a value for the small RNAs, use the recommended amount (5-10 µg) for small RNA library preparation. For samples that are too low to detect, we recommend using the entire sample (up to 11.5 µl) as the input for library preparation.
    10. If isolating DNA: DNA is now ready for quantification and submission to sequencing facility.
      1. Quantification: 2 µl + Qubit dsRNA HS Assay Kit.

Part III. Multiplexed small RNA libraries preparation
Notes:

  1. For small RNAs from Total RNA: Recommend 5-10 ng of small RNAs (i.e. isolated from 50 µg of Total RNA).
  2. Protocol has been successfully used to multiplex 2, 3, 4, 5, 6, 10 or 12 samples in one flow-cell lane.
  3. Typical levels of adaptor-adaptor contamination in the Illumina data are <1%.
  4. Duration of this step:
    1. Step 1 is an overnight step.
    2. Steps 2-7 can be performed in 1 day, with step 8 plus DE81 elution on the second day.
  5. Left over RNA ligation products and cDNA products can be saved at -20 ºC to be used to create additional DNA amplicons if the final concentration is insufficient for sequencing.
  6. Mix and spin refers to a brief vortex followed by a brief centrifugation.

  1. 3' adaptor ligation (ATP-independent)
    1. Set up the ligation reaction in a sterile, nuclease free 200 µl PCR tube on ice for each sample using the following:
      Reagent (stock concentration)
      Volume (final concentration)
      3’ Adaptor (10 µM)
      1.5 µl (1.3 µM)
      RNA (variable)
      Variable (10 ng)
      H2O
      Final volume: 11.5 µl
    2. Mix and spin samples.
    3. Incubate the tubes in a pre-heated thermal cycler at 70 °C for 2 min and then place the tubes on ice.
      Note: It is very important to keep the mixture on ice after the 70 °C incubation to prevent secondary structure formation.
    4. Pre-cool the thermocycler to 16 °C.
    5. Prepare the following master mix on ice (1x):
      Reagent (stock concentration)
      Volume (final concentration)
      Ligation buffer (10x)
      1.5 µl (1x)
      RNaseOUT (40 U/µl)
      1 µl (40 U)
      T4 RNA ligase 2, truncated, K227Q (200 U/µl)
      1 µl (200 U)
    6. Add 3.5 µl of the master mix to each tube. Mix and spin.
    7. Incubate the tubes in the pre-cooled thermocycler at 16 °C overnight (15-18 h). See note B in Notes section.

  2. 3' adaptor de-adenylation
    1. To each Ligation reaction, add 1 µl of De-adenylase (20 U/µl). Mix and spin.
    2. Incubate the tube in the thermocycler at 30 °C for 15 min.

  3. 3' adaptor digestion
    1. To each Ligation reaction, add 1 µl of Exonuclease VII (10 U/µl). Mix and spin.
    2. Incubate the tube in the thermocycler at 37 °C for 15 min.

  4. 5' adaptor ligation (ATP-dependent)
    1. Aliquot the required amount of the 5’ RNA adaptor (1 µl/reaction) into a thin-walled 200 µl PCR tube then incubate at 70 °C for 2 min and chill on ice.
    2. In this tube, prepare a Master Mix on ice using the following (1x):
      Reagent (stock concentration)
      Volume (final concentration)
      5’ RNA adaptor (15 pmol/µl)
      1 µl (15 pmol)
      dATP (10 mM)
      1 µl (0.48 mM)
      T4 RNA ligase (5 U/µl)
      1 µl (5 U)
      H2O
      1 µl
    3. Add 4 µl of the master mix to each ligation tube. Mix and spin.
    4. Incubate at 28 °C for 1 h, and then place the tube on ice.

  5. cDNA synthesis
    1. Label a new tube for each sample. Transfer 6 µl of the ligation product to the new tube.
    2. Prepare a master mix on ice using the following (1x):
      Reagent (stock concentration)
      Volume (final concentration)
      RT-PCR primer (100 µM)
      2.5 µl (5 µM)
      dNTPs (10 mM)
      2.5 µl (0.5 mM)
      H2O
      18 µl
    3. Add 23 µl of the master mix to each tube. Mix and spin.
    4. Heat tubes to 65 °C for 5 min in thermocycler. Cool on ice for at least 2 min.
    5. Prepare a second master mix with the following (1x):
      Reagent (stock concentration)
      Volume (final concentration)
      RT buffer (10x)
      5 µl (1x)
      MgCl2 (25 mM)
      10 µl (5 mM)
      DTT (100 mM)
      5 µl (10 mM)
      RNaseOUT (40 U/µl)
      1 µl (40 U)
    6. Add 21 µl of the master mix to each sample (final volume in each tube: 50 µl). Mix and spin.
    7. Incubate tubes on bench top for 2 min to allow for primer-RNA hybridization.
    8. Add 1 µl SuperScript III RT polymerase (200 U/µl) to each tube. Mix and spin. Transfer to thermocycler:
      a. Polymerization reaction: 50 min at 50 °C.
      b. Inactivate reaction: 5 min at 85 °C.
    9. Cool the tubes to 4 °C, spin them briefly, and then add 1 µl of RNase H (2 U). Mix well and spin. Transfer to thermocycler:
      a. 37 °C for 20 min.
    10. Cool samples on ice.

  6. Linear PCR Amplification
    1. Label a new set of tubes. Transfer 25 µl of the cDNA reaction product to the new tubes.
    2. Prepare a master mix on ice using the following (1x):
      Reagent (stock concentration)
      Volume (final concentration)
      Phusion HF buffer (5x)
      10 µl (1x)
      5’ PCR primer (25 µM)
      0.5 µl (0.25 µM)
      3’ indexed PCR primer (25 µM)
      0.5 µl (0.25 µM)
      dNTP Mix (10 mM)
      0.5 µl (0.1 mM)
      Phusion polymerase (2 U/µl)
      0.5 µl (1 U)
      H2O
      13 µl
    3. Aliquot 25 µl of the master mix to each tube. Mix and spin.
    4. Recommended PCR conditions:
      1. 30 sec at 98 °C
      2. Up to 17 cycles of: 10 sec at 98 °C; 30 sec at 60 °C; 15 sec at 72 °C
      3. 10 min at 72 °C
      4. Hold at 4 °C

  7. Verification Gel
    1. Pour a 2% agarose gel with 20-well comb. Mix 5 µl of PCR product + 5 µl of loading dye (preferably Orange G or a dye that will not obscure a band at ~100 bp). Load all 10 µl on gel and run at 80-85 V for ~20 min. Include a DNA ladder that has a 100 bp band (e.g. 50 bp step ladder).
    2. While agarose gel is running, clean up the PCR reactions using Qiagen QIAquick PCR Purification Kit.
      1. Add 5 µl of 3 M NaOAc (pH 5.5) to PCR sample + buffer PB to adjust the pH.
      2. Elute with 30 µl of EB.
    3.  Visualize agarose gel - the desired band should be just above 100 bp (Figure 4). Expected size of successful amplicons = 112-120 bp (5’Adaptor: 49 bp, 3’Adaptor: 45 bp, small RNA: 18-26 bp) (Figure 4A). Adaptor-adaptor products are 94 bp (Figure 4B).


      Figure 4. Agarose check gel with 50 bp step ladder. A. Amplicon band is the desired size, above 100 base pairs. B. Sample band is smaller than expected due to the excess of adaptor-adaptor product.

  8. Purification gel
    1. Prepare 6% native PAGE/0.5x TBE gel with 15-well comb by combining the following:
      Reagent (stock concentration)
      Volume (final concentration)
      Acrylamide: bis 37.5:1 solution (30%)
      6 ml (6%)
      TBE (10x)
      1.5 ml (0.5x)
      H2O
      22.2 ml
      1. Mix the above together thoroughly by inversion.
      2. Add the remaining reagents:
        Reagent (stock concentration)
        Volume (final concentration)
        TEMED
        33 µl
        APS (10%)
        240 µl (0.08%) *Mix thoroughly by inversion
      3. Pour gel into assembled glass plates and allow to polymerize for at least 30 min.
    2. Prepare samples: Add BPB dye in a 2 (sample):1 (BPB) ratio (i.e. 30 µl of sample to 15 µl loading buffer).
      1. Load 15 µl/lane (for a total of 3 lanes / sample).
      2. This is to prevent overloading one lane, which leads to smearing.
    3. Use 1 µl/lane of 25 bp step ladder or 1 µl/lane of 50 bp step ladder for markers in a final volume of 15 µl.
    4. Run gel at 100 V until BPB runs off the bottom edge of the gel (~2.5 to 3 h).
    5. Stain with SybrGold (35 µl in 100 ml 0.5x TBE) for at least 10 min. Carefully excise the bands corresponding to DNA amplicons (Figure 5), avoiding any adaptor-adaptor bands. Use a different razor for each sample. Clean up DNA using DE81 transfer method (see section B). When multiple lanes are used for one sample, transfer all lanes onto the same piece of DE81 paper.


      Figure 5. DNA amplicon purification gel. Desired amplicons are indicated (above 100 bp marker). Undesired adaptor-adaptor products are also present but at a reduced level compared to the DNA amplicons.

  9. Sample multiplexing
    1. In order to submit small RNA libraries to a sequencing center as a multiplexed sample, the individual samples must be mixed together. After quantification of each library preparation using the Qubit dsDNA HS Assay Kit, calculate the approximate nanomolar (nM) concentration of each sample. The following formula can be used:

      Libraries can now be mixed together in equal nanomolar amounts. The final concentration of the mixed sample will depend on the specific requirements of the sequencing center.

Notes

  1. In Carbonell et al. (2012), small RNA libraries were constructed from Total RNA and small RNA that co-immunoprecipitated with Arabidopsis thaliana ARGONAUTE1. For co-immunoprecipitated small RNA, the isolation of small RNA by gel electrophoresis (procedure A) was skipped, and samples were used directly in procedure C.
  2. This protocol has been used to prepare multiplexed small RNA libraries from fungi (unpublished results) and should be valid for other organisms. We recommend reducing the 3’ adaptor ligation incubation time to 1-2 h for samples from organisms with small RNAs that lack 2’-O-methylation at their 3’ end (Munafó and Robb, 2010).
  3. After sequencing, data should be available in FASTQ format. In order to use the small RNA data in downstream applications, the raw FASTQ data need to be de-multiplexed (identification of the sample index sequence) and 3’ adaptor trimmed (identification and removal of miRNA cloning linker 1). We provide the script LibParse.pl (https://github.com/carringtonlab/srtools) to processes small RNA data sets generated using the protocol described here. LibParse.pl outputs small RNA sequences in FASTA format with read counts for each unique sequence. Additional scripts in the SRTOOLS repository are designed to utilize LibParse.pl output. Alternatively, the utility fastx_clipper from the FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/) can be used instead of LibParse.pl to output de-multiplexed and adaptor-trimmed small RNA in FASTQ format for broader compatibility with downstream applications.
    LibParse.pl example:
    LibParse.pl -f in.fastq -t fastq -o libparse.prefix -r 50 -a libparse.failed -l libparse.log -v -e CTGTAG -E CGATGT,GATCAC,CAGATG,TACGTT,TTACCA,ACTGTA,ATCACG,ACTTGT,GCCAAT,TGCTAG,CTTGTA,TCAGGC
    fastx_clipper example:
    fastx_clipper -Q33 -l 18 -i in.fastq -c -v -M 23 -a CTGTAGGCACCATCAATCGATGT -o out.I1.fastq
    fastx_clipper -Q33 -l 18 -i in.fastq -c -v -M 23 -a CTGTAGGCACCATCAATGATCAC -o out.I2.fastq
    fastx_clipper -Q33 -l 18 -i in.fastq -c -v -M 23 -a CTGTAGGCACCATCAATCAGATG -o out.I3.fastq
    fastx_clipper -Q33 -l 18 -i in.fastq -c -v -M 23 -a CTGTAGGCACCATCAATTACGTT -o out.I4.fastq
    fastx_clipper -Q33 -l 18 -i in.fastq -c -v -M 23 -a CTGTAGGCACCATCAATTTACCA -o out.I5.fastq
    fastx_clipper -Q33 -l 18 -i in.fastq -c -v -M 23 -a CTGTAGGCACCATCAATACTGTA -o out.I6.fastq
    fastx_clipper -Q33 -l 18 -i in.fastq -c -v -M 23 -a CTGTAGGCACCATCAATATCACG -o out.I7.fastq
    fastx_clipper -Q33 -l 18 -i in.fastq -c -v -M 23 -a CTGTAGGCACCATCAATACTTGT -o out.I8.fastq
    fastx_clipper -Q33 -l 18 -i in.fastq -c -v -M 23 -a CTGTAGGCACCATCAATGCCAAT -o out.I9.fastq
    fastx_clipper -Q33 -l 18 -i in.fastq -c -v -M 23 -a CTGTAGGCACCATCAATTGCTAG -o out.I10.fastq
    fastx_clipper -Q33 -l 18 -i in.fastq -c -v -M 23 -a CTGTAGGCACCATCAATCTTGTA -o out.I11.fastq
    fastx_clipper -Q33 -l 18 -i in.fastq -c -v -M 23 -a CTGTAGGCACCATCAATTCAGGC -o out.I12.fastq

Recipes

  1. 37.5:1 acrylamide:bis solution for 7M-Urea, 17%-polyacrylamide gel and 6% native PAGE/0.5x TBE gel
    300 g of acrylamide and 8 g of bisacrylamide dissolved in 300 ml pre-warmed deionized water. Once dissolved, adjust final volume to 1 L with warm deionized water. Filter sterilize with 0.45 µM bottle-top vacuum filter; wrap bottle in aluminum foil and stored at 4 °C.
  2. 10% ammonium persulfate (APS)
    Add 10 g of ammonium persulfate to 80 ml of deionized water
    Stir to dissolve
    Adjust final volume to 100 ml
    Syringe filter into 10 ml aliquots and store at -20 °C
  3. 2x RNA loading dye (50 ml)
    47.5 ml of 100% formamide
    125 µl of 10% SDS
    0.0125 g of bromophenol blue
    50 µl of 0.5 M EDTA
    2.3 ml of H2O
    Final concentration:
    Formamide: 95%
    SDS: 0.025%
    Bromophenol blue: 0.025%
    EDTA: 0.5 mM
  4. Low salt buffer (50 ml)
    0.5 ml of 1 M Tris-HCl (pH 7.6)
    1.0 ml of 5 M NaCl
    0.1 ml of 0.5 M EDTA
    48.4 ml of H2O
    Final concentration:
    Tris-HCl (pH 7.6): 10 mM
    NaCl: 100 mM
    EDTA: 1 mM
  5. High salt buffer (50 ml)
    0.5 ml of 1 M Tris-HCl (pH 7.6)
    10 ml of 5 M NaCl
    0.1 ml of 0.5 M EDTA
    0.526 g of L-Arginine
    39.4 ml of H2O
    Final concentration:
    Tris-HCl (pH 7.6): 10 mM
    NaCl: 1 M
    EDTA: 1 mM
    L-Arginine: 50 mM

Acknowledgments

The original version of this protocol was described in Carbonell et al. (2012). The updated version of the protocol was described in Carbonell et al. (2014). This work was supported by grants from the National Science Foundation (MCB-0956526, MCB-1231726) and National Institutes of Health (AI043288).

References

  1. Carbonell, A., Fahlgren, N., Garcia-Ruiz, H., Gilbert, K. B., Montgomery, T. A., Nguyen, T., Cuperus, J. T. and Carrington, J. C. (2012). Functional analysis of three Arabidopsis ARGONAUTES using slicer-defective mutants. Plant Cell 24(9): 3613-3629.
  2. Carbonell, A., Takeda, A., Fahlgren, N., Johnson, S. C., Cuperus, J. T. and Carrington, J. C. (2014). New generation of artificial MicroRNA and synthetic trans-acting small interfering RNA vectors for efficient gene silencing in Arabidopsis. Plant Physiol 165(1): 15-29.
  3. Dretzen, G., Bellard, M., Sassone-Corsi, P. and Chambon, P. (1981). A reliable method for the recovery of DNA fragments from agarose and acrylamide gels. Anal Biochem 112(2): 295-298.
  4. Munafo, D. B. and Robb, G. B. (2010). Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA. RNA 16(12): 2537-2552.

简介

高通量测序是探索植物中小RNA群体的强大工具。 来自Illumina测序系统的不断增加的输出允许多重多个样品,同时仍然获得足够的数据用于小RNA发现和表征。 在这里我们描述了一个协议,用于生成多重小RNA文库,用于在Illumina HiSeq系统单端,50碱基对运行的一个泳道中测序多达12个样品。 RNA连接酶用于将3'和5'接头添加至纯化的小RNA; 缺少小RNA分子(衔接子 - 衔接子产物)的连接产物被故意耗尽。 cDNA合成后,线性PCR步骤扩增DNA片段。 本文使用的3'PCR引物包括独特的6-核苷酸序列,以允许多达多达12个样品。

关键字:小核糖核酸, 高通量测序技术, 文库制备, 复用, sRNA序列

材料和试剂

  1. 低分子量凝胶基小RNA分离
    1. ssRNA标记(Takara Bio公司,目录号:3416)
    2. 17%聚丙烯酰胺试剂,7M尿素PAGE
      1. 37.5:1聚丙烯酰胺:双丙烯酰胺(参见配方)
      2. 尿素(Life Technologies,目录号:15505-050)
      3. TEMED(Life Technologies,目录号:15524-010)
      4. 10%过硫酸铵(Sigma-Aldrich,目录号:A3678-100G)(参见Recipes)
    3. 2x RNA加载染料(参见配方)

  2. 基于DE81的核酸纯化
    1. 玻璃纤维(康宁公司,目录号:988-10144)
    2. 超厚污渍纸(Bio-Rad Laboratories,目录号:170-3969)
    3. Whatman DE81离子交换纤维素色谱纸(Thermo Fisher Scientific,目录号:05-717-1A)
    4. GlycoBlue共沉淀剂(Life Technologies,目录号:AM9516)
    5. 100%乙醇
    6. 75%乙醇
    7. EB缓冲液(QIAGEN,目录号:19086)
    8. 用于定量纯化的小RNA的Qubit RNA HS测定试剂盒(Life Technologies,目录号:Q32852)
    9. 用于定量DNA扩增子的Qubit dsDNA HS测定试剂盒(Life Technologies,目录号:Q32854)
    10. 低盐缓冲液(见配方)
    11. 高盐缓冲液(见配方)

  3. 多重小RNA文库制备
    1. 8-Strip PCR薄壁,200μl管(Corning Incorporated,Axygen ,目录号:PCR-0208-CP-C)
    2. miRNA克隆接头1(/5'App/CTGTAGGCACCATCAAT/3'ddC /; 1nm)(Integrated DNA Technologies,目录号:11-04-03-05)
    3. 截短的K227Q突变T4RNA连接酶2(New England Biolabs,目录号:M0351L)
    4. 去腺苷酶(New England Biolabs,目录号:M0331S)
    5. 外切核酸酶VII(United States Biological,目录号:70082Z)
    6. RNA 5'衔接头(GUUCAGAGUUCUACAGUCCGACGAUC)(Illumina RA5,目录号:15013205)
    7. dATP(Life Technologies,目录号:55082)
    8. T4 RNA连接酶I(Life Technologies,目录号:AM2141)
    9. RT-PCR引物(ATTGATGGTGCCTACAG; 25nmol;脱盐)(Integrated DNA Technologies)
    10. SuperScript III(Life Technologies,目录号:18080051)
    11. Phusion高保真II(Thermo Fisher Scientific,目录号:F549L)
    12. 5'PCR引物(Illumina小RNA PCR引物2:AATGATACGGCGACCACCGACAGGTTCAGA-GTTCTACAGTCCGA)
    13. 3'标引的PCR引物I1-I12(100nmol; PAGE-纯化)(Integrated DNA Technologies) 注意: 下面的条形码(下划线序列)是反向互补的 最后的Illumina输出序列(参见方括号中的序列 每个引物)。
      I1 [CGATGT]:CAAGCAGAAGACGGCATACGA ACATCG ATTGATGGTGCCTACAG
      I2 [GATCAC]:CAAGCAGAAGACGGCATACGA GTGATCA TTGATGGTGCCTACAG
      I3 [CAGATG]:CAAGCAGAAGACGGCATACGA CATCTGA TTGATGGTGCCTACAG
      I4 [TACGTT]:CAAGCAGAAGACGGCATACGA AACGTAA TTGATGGTGCCTACAG
      I5 [TTACCA]:CAAGCAGAAGACGGCATACGA TGGTAAA TTGATGGTGCCTACAG
      I6 [ACTGTA]:CAAGCAGAAGACGGCATACGA TACAGT ATTGATGGTGCCTACAG
      I7 [ATCACG]:  CAAGCAGAAGACGGCATACGA CGTGAT ATTGATGGTGCCTACAG
      I8 [ACTTGT]:  CAAGCAGAAGACGGCATACGA ACAAGT ATTGATGGTGCCTACAG
      I9 [GCCAAT]:  CAAGCAGAAGACGGCATACGA ATTGGCA TTGATGGTGCCTACAG
      I10 [TGCTAG]:  CAAGCAGAAGACGGCATACGA CTAGCA ATTGATGGTGCCTACAG
      I11 [CTTGTA]:  CAAGCAGAAGACGGCATACGA TACAAG ATTGATGGTGCCTACAG
      I12 [TCAGGC]:  CAAGCAGAAGACGGCATACGA GCCTGA ATTGATGGTGCCTACAG
    14. QIAquick PCR纯化试剂盒(QIAGEN,目录号:28106)
    15. 焦碳酸二乙酯(DEPC)-H 2 O/RNase-free H 2 O
    16. DNA大小标记,适合检测〜120bp片段(50bp步骤 梯,Promega公司,目录号:G4521; 25 bp步梯, Promega Corporation,目录号:G4511)
    17. 3 M乙酸钠(NaOAc)(pH 5.5)
    18. 6%天然PAGE的试剂
      1. 37.5:1聚丙烯酰胺:双丙烯酰胺(参见配方)
      2. TEMED(Life Technologies,目录号:15524-010)
      3. 10%过硫酸铵(Sigma-Aldrich,目录号:A3678-100G)(参见Recipes)

设备

  1. 热循环仪
  2. 微量离心机:在室温和4℃下使用
  3. Fisher凝胶负载尖端(Thermo Fisher Scientific,目录号:02-707-182)
  4. 琼脂糖凝胶电泳系统(Bio-Rad Laboratories,Sub-Cell GT system)
  5. 聚丙烯酰胺凝胶电泳系统(Thermo Fisher Scientific,猫头鹰双凝胶垂直电泳系统)
  6. 65°C水浴
  7. 半干印迹系统(Sigma-Aldrich,半干印迹,目录号:Z340502)
  8. 加热块

程序

低分子量凝胶基小RNA分离
注意:

  1. 如果您已经纯化了小RNA(例如通过免疫沉淀),则不需要该步骤。 请参阅Notes部分中的注释A. 在这种情况下,请跳到C部分。
  2. 此步骤的持续时间:
    1. 凝胶制备和聚合:1+ h。
    2. 凝胶预运行时间:30分钟以上。
    3. 凝胶运行时间:4+ h,加载时间和染色。

  1. 制备含有7M尿素的0.5x TBE中的17%聚丙烯酰胺凝胶
    1. 在计划运行凝胶的前一天,组装玻璃板和盖子用Saran包装保护。
      1. 使用14厘米x 16厘米和1.5毫米间隔板。
      2. 清洁所有材料(板,垫片和梳子)。
        1. 用10%十二烷基硫酸钠(SDS)洗涤
        2. 用蒸馏水彻底冲洗。
        3. 用95%乙醇冲洗。
        4. 允许风干。
    2. 同时制备聚丙烯酰胺凝胶混合物(下图):密封外边缘 的板用2%琼脂糖(55℃)装配以防止泄漏。 密封   在几分钟内完成。
    3. 通过组合以下物质制备用于制备一个具有15孔梳的17%变性尿素-PAGE/0.5x TBE凝胶的混合物(30ml):
      试剂(贮存浓度)
      体积(最终浓度)
      丙烯酰胺:双37.5:1的溶液(30%) 17 ml(17%)
      尿素
      12.6g(7M)
      TBE(10x)
      1.5 ml(1x)
      17 ml(17%)
      尿素
      12.6g(7M)
      TBE(10x)
      1.5 ml(1x)
      ...
    4. Let stand on bench for ~10 min then transfer to an ice bath in the cold room (4 °C) for 15+ min.
    5. Add the remaining reagents:
      Reagent (stock concentration)
      Volume (final concentration)
      TEMED
      25 µl *Mix thoroughly by inversion
      APS (10%)
      150 µl (0.05%) *Mix thoroughly by inversion
    6. Pour the gel and allow it to polymerize for at least 30 min; should see beginning of polymerization within ~4 min.
    7. 装配您的电泳装置,并将运行缓冲液(0.5×TBE)加入室内和室外
    8. 用注射器冲洗孔; 冲洗掉玻璃板底部边缘的气泡
    9. 凝胶必须预运行(在180V)30-60分钟以除去所有痕量 APS(铵和过硫酸根离子),以分布/平衡任何 添加到运行缓冲液中的稳定因子或离子,和 以确保在样品运行期间恒定的凝胶温度。 预跑时,带的凹形前端的出现减少 凝胶。 如果需要,可以加载空白溶液:混合等量 DEPC-H 2 O和2x负载染料。 15μl/泳道就足够了。

    10. 准备RNA样品并运行凝胶
      *将样品体积保持在最小分辨率(≤50μl)。
      1. 使用2x RNA加样染料以最终浓度1x制备样品,标记,梯子和空白等体积。
      2. 将样品在65℃加热10分钟,立即在冰上淬灭。 短暂旋转以收集试管底部的样品。
      3. 加载样品。
        注意:   在装载样品前,用注射器清洁通道以除去 过量的尿素。 样品加载决定了您的最终质量 凝胶。 我们使用费歇尔凝胶加载提示加载小RNA凝胶。 加载 样品在井的最底部在平带中,没有三角形, blob或其他草率加载。
      4. 在0.5×TBE缓冲液中在180V下运行,直到溴酚蓝(BPB)染料到达凝胶底部(〜4至4.5小时)。
      5. 在0.5×TBE缓冲液中用溴化乙锭染色凝胶
      6. 参见下文,使用DE81纸从丙烯酰胺片段洗脱RNA
    11. 第二部分。 基于DE81的核酸纯化
      注意:此步骤的持续时间

      1. 切割凝胶切片和半干转移:40分钟以上。
      2. 洗脱(RNA):60+分钟(第1天); 沉淀1:过夜; 沉淀#2:2.5+ h。
      3. 洗脱(DNA):60+分钟(第1天); 沉淀#1:1+ h; 沉淀#2:2+ h。

      1. 准备洗脱管
        1. 从500微升离心管中取出盖子,戳一个孔   底部中心用25号针(图1A)。 标签   管
        2. 材料〜25-40毫克无菌玻璃棉在底部(不 通过孔)使用无菌牙签或移液管尖端。 将玻璃棉紧密地压在0.1毫升刻度标记的下面 管离开DE81纸和缓冲器的空间(图1B)
        3. 从2毫升微量离心管(图1C)取出盖,并放置准备的500微升管(图1D)内。


          图   1.洗脱管的制备。 A.500μl微量离心管 已经去除了帽并且在底部中心制造了一个孔。 B.玻璃棉 放置在管内并压实,为缓冲液留下空间 和DE81纸。 C.取出2ml微量离心管。 D. 准备500微升管放置在2毫升管内,并准备   使用。

      2. 核酸切除和转移
        1. 放置一个厚的Whatman纸,用0.5x TBE预浸泡,半干 吸墨纸 用试管或10ml移液管滚动以除去任何气泡
        2.  从凝胶中消除所需的片段(图2),并放置在一块 DE81纸用铅笔标记。 每个样品/重复应该 放在自己的DE81纸上。 DE81纸含有二乙基氨基乙基 (DEAE)纤维素官能团,其将结合核酸, 可以通过加入高盐缓冲液来逆转(Dretzen等人, 1981)。
          包括转移控制 - 即传输含有来自梯形泳道的条带的凝胶片段。


          图   2.用17%聚丙烯酰胺 - 尿素凝胶进行小RNA分离染色 溴化乙锭。 A.小RNA切除前的总RNA。 同样 泳道,切除小RNA(18-26nt)。 ssRNA标记大小 表示。

        3. 将纸+凝胶放在浓厚的Whatman纸上的吸纸器。
        4. 将第二块预浸泡的厚Whatman纸放在上面。
        5. 小心地滚动第二张Whatman纸的顶部以除去任何气泡。
        6. 在半干印迹中以400mAmp转移20分钟。
        7. 取出顶部的Whatman纸。
          1. 通过检查凝胶片段和DE81纸的验证转移 标记的转移控制,并排,在紫外透射仪上。   核酸不应在凝胶片段中可见(图3A),但是 应该在DE81纸上可见(图3B)。 转移ssRNA 标记可能难以可视化

            图3.验证 核酸转移。 A.概述了凝胶片段,没有核酸 是可见的。 B.可见核酸转移的DE81纸(50bp DNA步梯)。

          2. 而凝胶+纸仍然在底部   Whatman纸,用剃刀剪掉多余的DE81纸。 丢弃 凝胶切片,并仔细填塞DE81纸进入洗脱管 含玻璃棉。

      3. 核酸洗脱
        1. 彻底淹没DE81纸〜200微升(通常200-500微升)的低 盐缓冲液。 使用移液管尖端轻轻推动纸尽可能低,所以它   完全被缓冲区覆盖。
        2. 在台式微量离心机中以最大速度旋转30秒。 丢弃流通。
        3. 重复低盐缓冲液洗涤两次,共三次洗涤。
        4. 将上层柱/纸组件转移到一个清洁,标记的2毫升管,取下盖子
        5. 洗脱:
          1. 向纸中加入150μl高盐缓冲液,并将其浸没
          2. 在70°C孵育15分钟 - 使用箔覆盖管。 不要使用水浴。
          3. 在台式微量离心机中短暂(10秒)旋转,并保存流量。
        6. 在过滤器组件下面放置一个新的2 ml管,并重复步骤C5(洗脱)两次
        7. 乙醇沉淀:
          1. 合并三种洗脱液并加入20μg糖原(例如GlycoBlue)。
          2.  加入2.5体积100%乙醇并混合。 理想的降水量   装配在单个1.5ml微量离心管中。 使用150μl的高盐 缓冲液+〜1,150μl乙醇将适合于1.5ml微量离心机 管=更有效的降水
          3. -80°C沉淀:RNA样品过夜; DNA样品为1 + h
          4. 在冷冻微量离心机中以最大速度离心:
            1. RNA样品为35分钟
            2. DNA样品15分钟。
          5. 倒出乙醇,用75%乙醇洗涤沉淀
          6. 在冷藏微量离心机中以最大速度旋转5分钟
          7.  在干净的KimWipe 10上倒掉乙醇和倒置管 min。 如有必要,使用干净的KimWipe清除多余的液体 管内 - 避免接触(蓝色)颗粒
          8. 重悬 沉淀在40μlDEPC处理的水中(如果一个样品的多个沉淀, 在此步骤中将池沉淀物最终体积为40μl)。
        8. 需要第二次沉淀以除去用于下游酶促步骤的残余盐:
          1. 加入4μl3 M NaOAc。 加入100μl100%乙醇。 混合。 在-80℃下沉淀至少2小时(或过夜)
            1. 按照步骤C7d-g。
            2. 将最终的沉淀重悬在10-12μlDEPC-H 2 O用于RNA或10-12μl用于DNA的EB缓冲液中。
        9. 如果分离小RNA:RNA现在已经准备好作为模板 多重小RNA文库制备步骤(下文C节)。
          1. 定量:2微升+ Qubit RNA HS测定试剂盒。 对于样品 Qubit返回小RNA的值,使用推荐量 (5-10μg)用于小RNA文库制备。 对于样品也是 低检测,我们建议使用整个样品(高达11.5μl)as 图书馆准备的输入。
        10. 如果分离DNA:DNA现在可以进行定量并提交到测序设备。
          1. 定量:2μl+ Qubit dsRNA HS测定试剂盒

      第三部分。 复制小RNA文库制备
      注意:

      1. 对于来自总RNA的小RNA:推荐5-10ng小RNA(即从50μg总RNA中分离)。
      2. 协议已经成功用于在一个流动池道中多路复用2,3,4,5,6,10或12个样品。
      3. Illumina数据中适配器 - 适配器污染的典型水平为<1%。
      4. 此步骤的持续时间:
        1. 步骤1是隔夜的步骤。
        2. 步骤2-7可以在1天内进行,步骤8加第二天的DE81洗脱。
      5. 如果最终浓度不足以进行测序,则可以在-20℃保存RNA连接产物和cDNA产物,以用于产生另外的DNA扩增子。
      6. 混合和旋转是指短暂的离心,然后短暂离心
      1. 3'衔接子连接(不依赖ATP)
        1. 在无菌,无核酸酶的200μlPCR管中在冰上为每个样品使用以下设置连接反应:
          试剂(贮存浓度)
          体积(最终浓度)
          3'适配器(10μM)
          1.5μl(1.3μM)
          RNA(可变)
          变量(10 ng)
          H sub 2 O
          最终体积:11.5μl
        2. 混合和旋转样品。
        3. 将管在预热的热循环仪中在70℃下孵育2分钟,然后将管置于冰上。
          注意:在70°C孵育后将混合物保持在冰上以防止二级结构形成是非常重要的。
        4. 将热循环仪预冷至16°C
        5. 在冰上制备以下主混合物(1x):
          试剂(贮存浓度)
          体积(最终浓度)
          连接缓冲液(10x)
          1.5μl(1x)
          RNaseOUT(40 U /μl)
          1μl(40 U)
          T4 RNA连接酶2,截短的K227Q(200U /μl)
          1μl(200 U)
        6. 向每个管中加入3.5μl主混合物。 混合和旋转。
        7. 将管在预冷却的热循环仪中在16℃下孵育过夜(15-18小时)。 请参阅注释部分中的注释B.

      2. 3'适体去腺苷酸化
        1. 向每个连接反应中,加入1μl去腺苷酶(20U /μl)。 混合和旋转。
        2. 在30℃下在热循环仪中孵育管15分钟。

      3. 3'衔接子消化
        1. 向每个连接反应中,加入1μl外切核酸酶VII(10U /μl)。 混合和旋转。
        2. 孵育管在热循环仪在37℃15分钟。

      4. 5'衔接子连接(ATP依赖性)
        1. 等分所需的5'RNA适配器(1微升/反应)的量   薄壁200μlPCR管,然后在70°C孵育2分钟和冷却 在冰上
        2. 在该管中,使用以下(1x)在冰上制备主混合物:
          试剂(贮存浓度)
          体积(最终浓度)
          5'RNA衔接子(15 pmol /μl)
          1μl(15 pmol)
          dATP(10mM) 1μl(0.48mM)
          T4 RNA连接酶(5U /μl) 1μl(5 U)
          H sub 2 O
          1微升
        3. 加入4μl的主混合物到每个结扎管。 混合和旋转。
        4. 在28℃孵育1小时,然后将管置于冰上。

      5. cDNA合成
        1. 为每个样品标记一个新试管。 将6μl连接产物转移到新试管中。
        2. 使用以下(1x)在冰上制备主混合物:
          试剂(贮存浓度)
          体积(最终浓度)
          RT-PCR引物(100μM)
          2.5μl(5μM)
          dNTPs(10mM)
          2.5μl(0.5mM)
          H sub 2 O
          18微升
        3. 添加23微升的主混合物到每个管。 混合和旋转。
        4. 在热循环仪中将管热至65℃5分钟。 在冰上冷却至少2分钟。
        5. 使用以下(1x)准备第二个主混音:
          试剂(贮存浓度)
          体积(最终浓度)
          RT缓冲区(10x)
          5μl(1x)
          MgCl 2(25mM) 10μl(5 mM)
          DTT(100mM)
          5μl(10 mM)
          RNaseOUT(40 U /μl)
          1μl(40 U)
        6. 向每个样品(每个管中的最终体积:50μl)中加入21μl主混合物。 混合和旋转。
        7. 在台面上孵育管2分钟,以允许引物-RNA杂交
        8. 加入1μlSuperScript III RT聚合酶(200 U /μl)到每个管。 混合和旋转。 转移到热循环仪:
          一个。 聚合反应:50℃下50分钟 b。 灭活反应:在85℃下5分钟
        9. 将管冷却至4℃,短暂旋转它们,然后加入1μl的RNase H(2U)。 充分混合并旋转。 转移到热循环仪:
          一个。 37℃20分钟
        10. 在冰上冷却样品。

      6. 线性PCR扩增
        1. 标记一套新的管。 将25μlcDNA反应产物转移到新试管中
        2. 使用以下(1x)在冰上制备主混合物:
          试剂(贮存浓度)
          体积(最终浓度)
          Phusion HF缓冲液(5x)
          10μl(1x)
          5'PCR引物(25μM)
          0.5μl(0.25μM)
          3'引物PCR引物(25μM) 0.5μl(0.25μM)
          dNTP混合物(10mM) 0.5μl(0.1mM)
          Phusion聚合酶(2U /μl)
          0.5μl(1 U)
          H sub 2 O
          13微升
        3. 等分25μl主混合物到每个管。 混合和旋转。
        4. 推荐的PCR条件:
          1. 在98℃下30秒/
          2. 高达17个循环:在98℃10秒; 60℃下30秒; 在72℃下15秒/
          3. 72℃10分钟
          4. 保持在4°C

      7. 验证凝胶
        1. 倒入2%琼脂糖凝胶与20孔梳。 混合5μlPCR产物+ 5μl 的加载染料(优选橙G或不会使a染色的染料) 带〜100bp)。 加载所有10微升在凝胶上,并在80-85 V运行〜20分钟。 包括具有100bp条带(例如 50bp步进梯)的DNA梯状物。
        2. 当琼脂糖凝胶运行时,使用Qiagen QIAquick PCR纯化试剂盒清除PCR反应。
          1. 向PCR样品+缓冲液PB中加入5μl3M NaOAc(pH5.5)以调节pH
          2. 用30μlEB洗脱。
        3.  可视化琼脂糖凝胶 - 所需的条带应刚好在100 bp以上 (图4)。 成功扩增子的预期大小= 112-120bp (5'适配子:49bp,3'适配子:45bp,小RNA:18-26bp)(图4A)。 适配器 - 衔接头产物为94bp(图4B)

          图4.琼脂糖 检查具有50bp步梯的凝胶。 A.扩增子带是所需的大小, 高于100个碱基对。 B.样本带小于预期,由于   过量的适配器 - 适配器产品。

      8. 纯化凝胶
        1. 通过组合以下:用15孔梳子制备6%天然PAGE/0.5x TBE凝胶;
          试剂(贮存浓度)
          体积(最终浓度)
          丙烯酰胺:双37.5:1的溶液(30%) 6 ml(6%)
          TBE(10x)
          1.5 ml(0.5x)
          H sub 2 O
          22.2ml
          1. 通过倒置将上述混合在一起。
          2. 添加剩余试剂:
            试剂(贮存浓度)
            体积(最终浓度)
            TEMED
            33微升
            APS(10%)
            240μl(0.08%)*通过反转彻底混合
          3. 将凝胶倒入组装的玻璃板中,并使其聚合至少30分钟。
        2. 制备样品:以2(样品):1(BPB)比(即,30μl样品加入15μl上样缓冲液)加入BPB染料。
          1. 加载15μl/泳道(总共3泳道/样品)。
          2. 这是为了防止过载一个通道,这导致拖尾。
        3. 使用1μl/泳道的25 bp步进梯或1μl/泳道的50 bp步进梯标记,最终体积为15μl。
        4. 在100V下运行凝胶,直到BPB跑出凝胶的底部边缘(〜2.5至3小时)
        5. 用SybrGold(35μl在100ml 0.5×TBE中)染色至少10分钟。 小心地切除对应于DNA扩增子的条带(图5), 避免任何适配器适配器带。 每个使用不同的剃刀 样品。 使用DE81转移法清除DNA(见B部分)。 什么时候 多个泳道用于一个样品,将所有泳道转移到同一个样品上   一块DE81纸

          图5.DNA扩增子纯化凝胶。显示了所需的扩增子(高于100bp的标记)。 不需要 适配器 - 适配器产品也存在但处于降低的水平 相比DNA扩增子

      9. 样品多路复用
        1. 为了提交小RNA文库到测序中心作为 多个样品,各个样品必须混合在一起。 后   使用Qubit dsDNA HS定量每个文库制备物 测定试剂盒,计算近似的纳摩尔浓度(nM) 每个样品。 可以使用以下公式:

          图书馆现在可以   以等量的纳摩尔量混合在一起。 最终浓度 混合样品将取决于具体要求 测序中心。

      笔记

      1. 在Carbonell等人(2012)中,从与拟南芥(Arabidopsis thaliana)ARGONAUTE1共免疫沉淀的总RNA和小RNA构建小RNA文库。对于共免疫沉淀的小RNA,通过凝胶电泳(步骤A)跳过小RNA的分离,并且将样品直接用于方法C中。
      2. 这个协议已被用于从真菌(未发表的结果)制备多路复用小RNA文库,应该适用于其他生物。我们建议将来自在其3'末端缺乏2'-O-甲基化的小RNA的生物体的样品的3'接头连接孵育时间减少至1-2小时(Munafó和Robb,2010)。
      3. 测序后,数据应以FASTQ格式提供。为了在下游应用中使用小RNA数据,原始FASTQ数据需要解复用(鉴定样品索引序列)和3' (miRNA克隆接头1的鉴定和去除)。我们提供了脚本LibParse.pl( https://github.com/carringtonlablabs/srtools )来处理小型RNA数据集使用此处描述的协议生成。 LibParse.pl输出FASTA格式的小RNA序列,每个独特序列的读数。 SRTOOLS存储库中的其他脚本旨在利用LibParse.pl输出。或者,来自FASTX工具包的实用程序fastx_clipper( http://hannonlab.cshl.edu/fastx_toolkit/)可用于替代LibParse.pl,以FASTQ格式输出解复用和适配器修剪的小RNA,用于与下游应用程序更广泛的兼容性。
        LibParse.pl示例:
        LibParse.pl -f in.fastq -t fastq -o libparse.prefix -r 50 -a libparse.failed -l libparse.log -v -e CTGTAG -E CGATGT,GATCAC,CAGATG,TACGTT,TTACCA,ACTGTA,ATCACG, ACTTGT,GCCAAT,TGCTAG,CTTGTA,TCAGGC
        fastx_clipper示例:
        fastx_clipper -Q33 -l 18 -i in.fastq -c -v -M 23 -a CTGTAGGCACCATCAATCGATGT -o out.I1.fastq
        fastx_clipper -Q33 -l 18 -i in.fastq -c -v -M 23 -a CTGTAGGCACCATCAATGATCAC -o out.I2.fastq
        fastx_clipper -Q33 -l 18 -i in.fastq -c -v -M 23 -a CTGTAGGCACCATCAATCAGATG -o out.I.3fastq
        fastx_clipper -Q33 -l 18 -i in.fastq -c -v -M 23 -a CTGTAGGCACCATCAATTACGTT -o out.I4.fastq
        fastx_clipper -Q33 -l 18 -i in.fastq -c -v -M 23 -a CTGTAGGCACCATCAATTTACCA -o out.I5.fastq
        fastx_clipper -Q33 -l 18 -i in.fastq -c -v -M 23 -a CTGTAGGCACCATCAATACTGTA -o out.I6.fastq
        fastx_clipper -Q33 -l 18 -i in.fastq -c -v -M 23 -a CTGTAGGCACCATCAATATCACG -o out.I7.fastq
        fastx_clipper -Q33 -l 18 -i in.fastq -c -v -M 23 -a CTGTAGGCACCATCAATACTTGT -o out.I8.fastq
        fastx_clipper -Q33 -l 18 -i in.fastq -c -v -M 23 -a CTGTAGGCACCATCAATGCCAAT -o out.I.9fastq
        fastx_clipper -Q33 -l 18 -i in.fastq -c -v -M 23 -a CTGTAGGCACCATCAATTGCTAG -o out.I10.fastq
        fastx_clipper -Q33 -l 18 -i in.fastq -c -v-M 23 -a CTGTAGGCACCATCAATCTTGTA -o out.I11.fastq
        fastx_clipper -Q33 -l 18 -i in.fastq -c -v-M 23 -a CTGTAGGCACCATCAATTCAGGC -o out.I12.fastq

      食谱

      1. 37.5:1丙烯酰胺:双溶液用于7M-尿素,17%聚丙烯酰胺凝胶和6%天然PAGE/0.5x TBE凝胶。
        300g丙烯酰胺和8g双丙烯酰胺溶解在300ml预热的去离子水中。 一旦溶解,用温去离子水调节终体积至1L。 用0.45μM瓶顶真空过滤器过滤灭菌; 用铝箔包裹并在4℃下储存
      2. 10%过硫酸铵(APS)
        将10克过硫酸铵加到80毫升去离子水中 搅拌溶解
        将最终体积调整为100 ml
        注射器过滤器成10毫升等分,存储在-20°C
      3. 2x RNA加载染料(50ml) 47.5ml 100%甲酰胺 125μl10%SDS
        0.0125g溴酚蓝
        50μl0.5M EDTA
        2.3ml H 2 O 2 / 最终浓度:
        甲酰胺:95%
        SDS:0.025%
        溴酚蓝:0.025%
        EDTA:0.5mM
      4. 低盐缓冲液(50ml)
        0.5ml 1M Tris-HCl(pH7.6)
        1.0ml 5M NaCl
        0.1ml 0.5M EDTA
        加入48.4ml H 2 O 2 / 最终浓度:
        Tris-HCl(pH7.6):10mM NaCl:100mM
        EDTA:1mM
      5. 高盐缓冲液(50ml)
        0.5ml 1M Tris-HCl(pH7.6)
        10ml 5M NaCl 0.1ml 0.5M EDTA
        0.526克L-精氨酸 加入39.4ml H 2 O 2 / 最终浓度:
        Tris-HCl(pH7.6):10mM NaCl:1MH 2 / EDTA:1mM
        L-精氨酸:50mM

      致谢

      该协议的原始版本在Carbonell等人(2012)中描述。协议的更新版本在Carbonell等人(2014)中描述。这项工作是由国家科学基金会(MCB-0956526,MCB-1231726)和国立卫生研究院(AI043288)的拨款支持。

      参考文献

      1. Carbonell,A.,Fahlgren,N.,Garcia-Ruiz,H.,Gilbert,K.B.,Montgomery,T.A.,Nguyen,T.,Cuperus,J.T.and Carrington, 使用切片缺陷突变体对三种拟南芥 ARGONAUTES的功能分析。 a> 植物细胞 24(9):3613-3629
      2. Carbonell,A.,Takeda,A.,Fahlgren,N.,Johnson,S.C.,Cuperus,J.T.and Carrington,J.C。(2014)。 新一代人工微RNA和合成的反式作用小干扰RNA载体,用于有效的基因沉默拟南芥 植物生理 165(1):15-29。
      3. Dretzen,G.,Bellard,M.,Sassone-Corsi,P.and Chambon,P。(1981)。 从琼脂糖和丙烯酰胺凝胶中回收DNA片段的可靠方法 Anal Biochem 112(2):295-298。
      4. Munafo,D.B.and Robb,G.B。(2010)。 优化用于从小RNA产生cDNA的代表性库的酶反应条件。 RNA 16(12):2537-2552。
      • 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。
      Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
      引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
      1. Gilbert, K. B., Fahlgren, N., Kasschau, K. D., Chapman, E. J., Carrington, J. C. and Carbonell, A. (2014). Preparation of Multiplexed Small RNA Libraries From Plants. Bio-protocol 4(21): e1275. DOI: 10.21769/BioProtoc.1275.
      2. Carbonell, A., Takeda, A., Fahlgren, N., Johnson, S. C., Cuperus, J. T. and Carrington, J. C. (2014). New generation of artificial MicroRNA and synthetic trans-acting small interfering RNA vectors for efficient gene silencing in Arabidopsis. Plant Physiol 165(1): 15-29.
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