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Individual-nucleotide-resolution UV Cross-linking and Immunoprecipitation (iCLIP) of UPF1
UPF1蛋白单核苷酸分离紫外线交联和免疫沉淀(iCLIP)   

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Abstract

The fate of mRNA, in particular its stability, localization and rate of translation is regulated by RNA binding proteins assembling to messenger ribonucleoprotein (mRNP) complexes. To investigate the transcriptome-wide RNA binding sites of UPF1, the core factor of nonsense-mediated mRNA decay (NMD), we performed individual-nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP) (Zund et al., 2013) followed by high-throughput sequencing. The presented protocol is optimized to investigate the RNA-binding sites of UPF1 and is based on previously described studies (Konig et al., 2010; Konig et al., 2011; Hafner et al., 2010). We want to thank the Group of Mihaela Zavolan (Swiss Institute of Bioinformatics, Basel, Switzerland) and Jernej Ule (Medical Research Council Laboratory of Molecualar Biology, Cambridge, UK) for technical support in setting up these experiments.

Materials and Reagents

  1. Hela cells
  2. DMEM (powder, high glucose) (Life Technologies, catalog number: 52100-039 )
  3. Fetal Calf Serum (FCS) (BioConcept, Amimed, catalog number: 2-01F30-I )
  4. Penicillin-Streptomycin solution (P/S) (1 unit/ml) (BioConcept, Amimed, catalog number: 4-01F00-H )
  5. Ice
  6. Trypsin-EDTA (T/E) (BioConcept, Amimed, catalog number: 5-5iF00-H )
  7. Liquid nitrogen
  8. DEPC BioChemica (AppliChem GmbH, catalog number: A0881, 0250 )
  9. 100x HaltTM Protease Inhibitor Cocktail (Thermo Fisher Scientific, catalog number: 1861279 )
  10. RNase I (cloned) (100 U/µl) (Life Technologies, Ambion®, catalog number: AM2294 )
  11. NaCl
  12. Turbo DNase (Life Technologies, Ambion®, catalog number: AM2238 )
  13. Goat anti-RENT1 antibody (Bethyl Laboratories, catalog number: A300-038A ) (UPF1 is also named RENT1.)
  14. Phenol
  15. Chloroform
  16. Isoamyl alcohol
  17. Dynabeads® Protein G (Life Technologies, catalog number: 10004D )
  18. Alkaline Phosphatase Calf Intestinal (CIP) (New England Biolabs, catalog number: M0290S )
  19. T4 RNA Ligase (10 U/µl) (Thermo Fisher Scientific, catalog number: EL0021 )
  20. RNasin (Dundee Cell Products, catalog number: RS3100 )
  21. 3’ RNA linker (5'-P-UGAGAUCGGAAGAGCGGUUCAG-Puromycin-3') (Microsynth AG; http://www.microsynth.ch/)
    Note: The RNA is delivered as 5 nmol aliquots, stored at -80 °C and dissolved in 25 µl MQ-water before usage.
  22. γ-P32-ATP (10 µCi/µl) (HARTMANN ANALYTIC GmbH, catalog number: SCP-401 )
  23. T4 Polynucleotide Kinase (10 U/µl) (Thermo Fisher Scientific, catalog number: EK0031 )
  24. 4x NuPAGE® LDS Sample Buffer (Life Technologies, catalog number: NP0007 )
  25. 20x NuPAGE® MOPS SDS Running Buffer (Life Technologies, catalog number: NP0001 )
  26. Prestained Protein Ladder (broad range) (New England Biolabs, catalog number: P7710S )
  27. GlycoBlueTM Coprecipitant (15 mg/ml) (Life Technologies, catalog number: AM9515 )
  28. Proteinase K (recombinat PCR grade) (Roche Diagnostics, catalog number: 03 115 887 001 )
  29. RT-primer (Microsynth AG)
    Oligo sequence: 5'-P-NNNNAACAGATCGGAAGAGCGTCGTGGATCCTGAACCGC-3'
  30. dNTP mix (10 mM each) (Thermo Fisher Scientific, catalog number: R0192 )
  31. AffinityScriptTM Multi-Temp RT (Agilent, catalog number: 600109-51 )
  32. 40% Acrylamide-Solution (mix 19:1, molecular biology grade) (AppliChem GmbH, catalog number: A3658 )
  33. Tetramethylethylendiamin (TEMED) (Sigma-Aldrich, catalog number: T22500-100ML )
  34. Ammonium persulfate (APS) (Sigma-Aldrich, catalog number: A3678-100G )
  35. PlusOne Repel-Silane ES (GE Healthcare, catalog number: 17-1332-01 )
  36. Low Molecular Weight DNA Ladder (New England Biolabs, catalog number: N3233L )
  37. SYBR® Gold Nucleic Acid Gel Stain (Life Technologies, catalog number: S11494 )
  38. CircLigaseTM II ssDNA Ligase (EpiCentre, catalog number: CL9025K )
  39. Cut-Oligo (5'- GTTCAGGATCCACGACGCTCTTCAAAA-3') (Microsynth AG)
  40. BamHI (New England Biolabs, catalog number: R0136S )
  41. P5_Solexa primer (Microsynth AG)
    Oligo sequence: 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3'
  42. P3_Solexa primer (Microsynth AG)
    Oligo sequence: 5'-CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT-3'
  43. Maximo Taq DNA 2X-Mastermix (GeneOn, catalog number: S114 )
  44. DMEM-/- (per 900 ml) (see Recipes)
  45. DMEM+/+ (per 500 ml) (see Recipes)
  46. Phosphate-bufferd saline (PBS) (pH 7.4) (see Recipes)
  47. Hypotonic gentle lysis buffer (pH 7.5) (RNase-free) (see Recipes)
  48. IP-wash buffer (RNase-free) (see Recipes)
  49. High salt wash buffer (pH 7.5) (RNase-free) (see Recipes)
  50. PNK buffer (pH 7.5) (RNase-free) (see Recipes)
  51. PNK buffer w/o DTT (pH 7.5) (RNase-free) (see Recipes)
  52. Proteinase K buffer (pH 7.5) (RNase-free) (see Recipes)
  53. TE buffer (pH 7.5) (RNase-free) (see Recipes)
  54. 10x Tris-borate buffer (TBE) (pH 8.0) (RNase-free) (see Recipes)
  55. Acidic phenol (see Recipes)
  56. Solution A (RNase-free) (see Recipes)
  57. Solution B (RNase-free) (see Recipes)
  58. Loading buffer for PAA-urea gel (pH 8.0) (RNase-free) (see Recipes)
  59. 10x DNA loading buffer (see Recipes)
  60. DEPC treated water/buffer (see Recipes)
  61. 3 M NaOAc (pH 4.6 and 5.5) (RNAse-free) (see Recipes)
  62. RNA ligation mix (see Recipes)
  63. Kinase mix (see Recipes)
  64. CircLigation mix (see Recipes)
  65. Oligo annealing mix (see Recipes)
  66. PCR master mix (see Recipes)

Equipment

  1. Pure water system: PURELAB Priama (Prima 7) and PURELABULTRA (Ultra Genetic) (ELGA LabWater)
    Note: In this protocol pure water from the ELGA system is referred to as MQ-water.
  2. CO2 incubator (BINDER GmbH, model: 9140-0047 )
  3. Clear-viewTM Snap-Cap microtubes (1.5 ml, natural, low retention) (Sigma-Aldrich, catalog number: T4816-250EA )
  4. Multiply®-Pro 0.2 ml Biosphere® (Sarstedt AG, model: 72.727 )
  5. Filter tips (10 µl, 20 µl and 200 µl) (Axon Lab AG, catalog number: AL60X10 , AL60X20 , AL60X200 )
  6. Filter tips (1,250 µl) (Greiner Bio-One GmbH, catalog number: 7.750.261 )
  7. 15 cm tissue culture dishes (TPP Techno Plastic Products, catalog number: 93150 )
  8. Stratalinker 2400 (254 nm) (Stratagene®, catalog number: 400076 )
  9. PCR tube
  10. Cell lifter (TPP Techno Plastic Products, model: 99010 )
  11. Saran wrap
  12. Scotch
  13. GP Millipore Express® PLUS membrane (0.22 µm) (500 ml Funnel, 45 mm Neck Size) (EMD Millipore, catalog number: SCGPT05RE )
  14. DynaMagTM-2 magnet (Life Technologies, catalog number: 12321D )
  15. Water bath (temperature adjustable)
  16. Eppendorf centrifuge 5415R with rotor F45-24-11 (Eppendorf, model: 022621459 and 022636502 )
  17. CriterionTM Cell (Bio-Rad Laboratories, catalog number: 165-6001 )
  18. NuPAGE® Novex® 4-12% Bis-Tris Midi Gels (1.0 mmx, 12 + 2 well) (Life Technologies, catalog number: WG1401BOX )
  19. Midi Gel Adaptor (Life Technologies, model: WA0999 )
  20. iBlot® Gel Transfer Devise (EU) (Life Technologies, model: IB1001EU )
  21. iBlot® Transfer Stack (nitrocellulose, regular size) (Life Technologies, catalog number: IB3010-01 )
  22. 10 ml serological pipette
  23. POWER PAC 3000 (Bio-Rad Laboratories)
  24. Lab cycler gradient equipped with Thermoblock 96 (SensoQuest GmbH, models: 011-101 and 012-103 )
  25. Blotting paper (Genentech, catalog number: BP 002 46579 )
  26. FUJI screen
  27. FUJIFILM FLA-3000 phosphorimager
  28. Costar Spin-X® centrifugation column (Corning, model: 8161 )
  29. Vertical PAA-gel running apparatus for 180 x 120 mm gels with 1 mm thick spacers and combs (home made)
  30. Paper clips foldback (51 mm) (Lyreco, catalog number: 161.791 )
  31. Aluminium plate (90 x 175 mm, thickness 3 mm)
  32. UV desk (365 nm) (home made)
  33. 1 ml syringe (BD, PlastipakTM, catalog number: 300013 )

Procedure

  1. Cell culture and UV cross-linking
    1. Hela cells are cultivated in Dulbecco’s Modified Eagle Media (DMEM) supplemented with 100 U/ml Penicillin, 100 µg/ml Streptomycin and 10% foetal calf serum in ten 15 cm diameter dishes until they are 80% confluent.
    2. The media is discarded and cells are covered with 12 ml ice-cold PBS buffer.
    3. The cells are subjected to 150 mJ/cm2 UV-C light. During the irradiation the dishes are placed on ice.
    4. After irradiation the cells are covered with 10 ml fresh ice-cold PBS and scraped off using a cell lifter.
    5. The cells are sedimented in two 50 ml falcon tubes by centrifugation (4 °C, 200 x g, 5 min), shock frozen in liquid nitrogen and stored until usage at -80 °C.
  2. Limited RNase I digestion and UPF1 immunoprecipitation
    1. The cells are thawed on ice and lysed in 7 ml hypotonic gentle lysis buffer for 20 min. The cell debris is removed by centrifugation (4 °C, 13,000 x g, 15 min). The supernatant is recovered and the NaCl concentration is adjusted to 150 mM by the addition of 350 µl of 3 M NaCl.
    2. For partial RNase digestion, RNase I is diluted 1:1,000 in hypotonic gentle lysis buffer and the cell lysate is treated with 70 µl of the RNase I dilution and 35 µl Turbo DNase with slight shaking in a 37 °C water bath for 3 min.
      Note: For each RNA binding protein subjected to iCLIP the RNase I digestion step has to be adjusted de novo in order to obtain 80-200 nt long RNA pieces bound to the protein of interest, suitable for cDNA library preparation and subsequent high-throughput sequencing (see Notes).
    3. The RNase I treated lysate is rested on ice for 3 min.
    4. 28 µl of goat anti-RENT1 antibody is added and rotated head over tail at 4 °C for 1 h.
    5. 240 µl of Dynabeads® Protein G are washed twice with 1 ml PBS buffer and once with 1 ml hypotonic gently lysis buffer. To collect the magnetic Dynabeads® the DynaMagTM-2 Magnet rack is used.
    6. After 1 h pre-incubation of the lysate with UPF1/RENT1 antibody the washed beads are added and incubated for an additional hour at 4 °C.
    7. Remove the cell lysate and wash the beads three times with IP wash buffer.
  3. Dephosphorylation of the RNA followed by the ligation of the 3’ RNA-linker
    1. To dephosporylate the RNA pieces covalently bound to UPF1, the Dynabeads® are resuspended in 60 µl dephosporylation mix [55.25 µl MQ-water, 6.5 µl 10x NEBuffer 3, 3.25 µl Alkaline Phosphatase Calf Intestinal (CIP)] and incubated at 37 °C for 20 min.
    2. The beads are washed twice with 1 ml high salt wash buffer for stringent washing and twice with 1 ml PNK buffer.
    3. After the last wash step the supernatant is carefully removed and the beads are incubated in 50 µl of RNA ligation mix. The over night ligation of the 3’ RNA linker is performed in a 0.2 ml PCR tube placed at 16 °C in a PCR machine. The lid is kept at constant 50 °C to prevent condensation of the sample.
  4. Radiolabeling of the RNA and isolation of the RNA-UPF1 complex
    1. The samples is washed twice with 1 ml PNK buffer and the RNA is radioactively labelled by incubating with 60 µl of kinase mix at 37 °C, shaking at 800 rmp for 45 min.
    2. The radiolabeled sample bound to the beads is washed four times with PNK buffer w/o DTT (The radioactive buffer after the second wash step is kept and used for the hot positioning marks in step 4i.).
    3. The protein-RNA complexes are eluted from the beads by cooking at 90 °C for 5 min in 45 µl 1x NuPAGE® LDS Sample Buffer.
    4. The gel running-apparatus is assembled using a BioRAD criterion chamber, a midi gel adaptor and a pre-casted NuPAGE® Novex® 4-12% Bis-Tris midi gel. The running chamber is filled with 500 ml 1x NuPAGE® MOPS SDS Running Buffer.
    5. The eluted protein-RNA sample (40 µl) is loaded on the NuPAGE® Novex Gel next to 6 µl prestained protein ladder and separated at 200 V for 45-60 min until the bromophenol blue running front is about to leave the gel.
    6. Disassemble the gel device and cut off the running front marked by the bromophenol blue band. This lower part of the gel contains the non-incorporated γ-P32-ATP and is disposed of as solid radioactive waste.
    7. The protein-RNA adducts are transferred to nitrocellulose membrane using the iBlot system (for detailed instruction consult the tutorials provided by the manufacturer).
    8. After the 7 min transfer procedure the transfer stacks are disassembled and disposed of as solid radioactive waste.
    9. The nitrocellulose membrane is wrapped in saran wrap and three positioning marks are attached at the edge of the membrane [1 µl of the hot PNK buffer put aside in step 4b is spread on a small blotting paper strip (1.5 x 8 mm) and fixed on the saran wrap with a small piece of Scotch. These marks are important to properly align the autoradiograph in step 4k to excise and isolate the UPF1-mRNA complex from the nitrocellulose membrane.].
    10. For the detection of the P32-labelled RNA-UPF1 complex on the nitrocellulose membrane a FUJI screen is exposed for 1 h and analysed on a FUJIFILM FLA-3000 phosphorimager.
    11. The autoradiograph of the membrane is printed in real seize format and aligned with the nitrocellulose membrane with help of the positioning markers. The section of the nitrocellulose membrane containing the RNA-UPF1 complex is excised and ground into small pieces using a 1,250 µl pipette tip.
    12. The RNA is isolated by incubating the membrane pieces with 200 µl proteinase K buffer supplemented with 20% (v/v) proteinase K at 50 °C for 1h.
    13. The elution is repeated to maximise the elution efficiency.
    14. The pooled elution fractions (final volume 400 µl) are filtered (spun at 2,000 x g for 2 min) using a costar Spin-X® centrifugation column.
  5. Phenol/chloroform extraction and RNA precipitation
    1. Add one volume of acidic phenol:chloroform:isoamylalcohol (25:24:1) to the filtered RNA sample and shake at 1,000 rpm (not vortex) for 5 min.
    2. Spin the sample (16,000 x g, 4 °C, 10 min) and transfer the aqueous phase (350 µl) into a fresh tube.
    3. Add 35 µl 3 M NaOAc (pH 4.6), 2 µl GlycoBlue Coprecipitant, 400 µl isopropanol; mix and precipitated the RNA over night at -20 °C.
    4. The RNA is precipitated by centrifugation (16,000 x g, 4 °C, 15 min), washed with 1 ml 70% EtOH and dissolved in 5.5 µl MQ-water.
  6. Reverse transcription
    1. Transfer the sample into a 0.2 ml PCR tube.
    2. 0.5 µl RNasin, 0.5 µl of dNTPs (10 mM each) and 0.5 µl RT-primer (0.5 pmol/µl) are added to the RNA and heated to 70 °C for 5 min (in a PCR thermo cycler).
    3. After slowly cooling the sample to 25 °C (temperature ramp: 1.8 °C/min), 3 µl of the following RT mix (1 µl 10x AffinityScript RT buffer, 1 µl 0.1 M DTT, 1 µl Affinity Script Multi-Temp RT) is added.
    4. Continue the reverse transcription with the following thermal program:
      25 °C   
      15 min
      50 °C
      60 min
      4 °C   
      hold
    5. The cDNA is transferred to 1.5 ml test tube, precipitated by adding 90 µl TE buffer, 0.5 µl GlycoBlue Coprecipitant, 10 µl NaOAc (pH 5.5), 250 µl 100% EtOH and a subsequent incubation at -20 °C for over night.
    6. The cDNA is pelleted by centrifugation (4 °C, 16,000 x g, 15 min).
    7. The pellet is washed with 70% EtOH.
    8. Resuspend the cDNA in 10 µl loading buffer for PAA-urea gels and cook at 70 °C for 5 min.
    9. Mix 2 µl low molecular weight marker with 2 µl 6x loading buffer and 8 µl loading buffer for PAA-urea gels (do not cook the marker).
  7. Cast 6% TBE-urea gel
    1. Wash and dry the glass plates (180 x 120 mm) carefully.
    2. Apply PlusOne repel-silane ES with a soft paper towel to the inner surface of the glass plates.
    3. Assemble the glass plates and the spacers (1 mm thickness) and fix the setup with two file clips centred on the spacers. Lay the assembled glass plates on an empty tip box. Casting the gel in horizontal position makes sealing of the boarders redundant.
    4. Prepare 25 ml 6% gel solution by mixing 7.5 ml solution A with 17.5 solution B. Start the polyimerization by adding 125 µl 10% APS and 12.5 µl TEMED.
    5. The gel solution is applied with a 10 ml serological pipette to edge of the glass plate on the side where the comb will be inserted. Surface tension and slight tapping on the glass plate ensures an even and bubble-free distribution of the gel solution.
    6. Introduce the comb and fix the glass plates with additional file clips.
    7. Polymerize the gel for 1 h.
  8. Run 6% TBE-urea gel
    1. Clamp the gel assembled with an aluminium plate into the gel running chamber (The 90 x 175 x 3 mm aluminium plate ensures even heat distribution and therewith even running of the samples in the gel.) and fill the apparatus with 1x TBE buffer.
    2. Wash the slots carefully with a syringe.
    3. Pre-run the gel (10 W, 30 min).
    4. Wash the slots again.
    5. Load the low molecular weight DNA ladder and the cDNA sample leaving 2-3 empty lanes in between.
    6. Run the gel (5 W, 70 min) until the bromophenol blue band is 2 cm above the bottom of the gel.
  9. Isolate cDNA
    1. Cut the gel in order to separate the lane containing the size ladder from the lane containing the sample.
    2. Stain the marker-containing gel part with 1x SYBR-gold in 1x TBE buffer (for 15 min) and wrap the part of the gel containing the sample in saran wrap.
    3. Visualize the DNA ladder on a 365 nm UV-desk and copy the position of the size ladder on a transparency.
    4. With help of the transparency showing the size ladder (placed next to the unstained gel part) excise the portion of the gel containing the cDNA covering a size range form 150-300 nt.
    5. Crush the gel piece with a syringe plunger (from a 1 ml syringe) and extract the cDNA with 400 µl TE buffer (shaking at 2,000 rpm, 37 °C, 2 h).
    6. Filter the supernatant with a Costar Spin-X® centrifugation column (2 min, 2,000 x g).
    7. Precipitate the cDNA by adding 1 µl GlycoBlue Coprecipitant, 40 µl NaOAc (pH 5.5) and 1 ml 100% EtOH and incubate at -20 °C for 1 h.
    8. Pellet the cDNA by centrifugation (4 °C, 16,000 x g, 15 min).
    9. Wash the pellet with 1 ml 70% EtOH.
    10. Dissolve the cDNA in 6.7 µl MQ-water.
  10. Introduction of the 5’ primer to the cDNA by circularization and subsequent linearization
    1. To perform the circularization add 1.5 µl CircLigation mix to the 6.7 µl cDNA followed by the incubation in a PCR thermo cycler at 60 °C for 1 h.
    2. To linearize the single stranded circular cDNA, a DNA cut-oligo is annealed to a region in the RT-primer generating a double-stranded DNA stretch harbouring a BamHI site.
      Prepare the oligo annealing mix and add 30 µl of it to the circular cDNA. The sample is heated to 90 °C and slowly cooled down to 25 °C (in PCR thermo cycler, 1 °C/min temperature ramp).
    3. To cleave the circular cDNA, 2 µl BamHI restriction enzyme is added and incubated at 37 °C for 45 min.
    4. Precipitate the linearized cDNA by adding 50 µl TE buffer, 0.5 µl GlycoBlue Coprecipitant, 10 µl NaOAc (pH 5.5) and 250 µl 100% EtOH prior to the incubation at -20 °C for over night.
    5. Pellet the linearized cDNA by centrifugation (4 °C, 13,000 x g, 15 min).
    6. Wash the pellet with 70% EtOH.
    7. The precipitated cDNA is dissolved in 50 µl MQ-water.
  11. PCR amplification of the cDNA
    The adapters needed for high-throughput Illumina sequencing are introduced by PCR using the P5_Solexa and the P3_Solexa primers.
    1. Pilot PCR
      In order to determine the number of cycles needed for a proper amplification of the cDNA library, a 100 µl test PCR is performed. To set up the test PCR, combine 10 µl cDNA, 40 µl PCR master mix and 50 µl Maximo Taq DNA mastermix and run the following PCR program:
      95 °C for 5 min
      34 cycles of (95 °C for 45 s, 50 °C for 80 s, 72 °C for 1 min)
      To analyze the intermediate PCR products after cycle 20, 22, 24, 26, 28, 30, 32 and 34, a 12 µl sample is taken and mixed with 3 µl 10x DNA loading buffer.
      Of each sample separate 7.5 µl on a 2.5% agarose gel to determine the number of cycles (Nthreshold) needed to generate just enough PCR product resulting in a band visible on the agarose gel. Overamplification should be avoided as it drastically reduces the complexity of the sequencing library.
    2. Preparative PCR
      For the preparative PCR amplification of the cDNA library four 100 µl PCR reactions are run with the following cycles:
      95 °C for 5 min
      Nthreshold cycles of (95 °C for 45 s, 50 °C for 80 s, 72 °C for 1 min)
      72 °C for 5 min
    3. Clean up the preparative PCR product
      The four PCR products are combined, mixed with 400 µl phenol:chlorofom:isoamylalcohol (25: 24: 1) and rotated head over tail at room temperature for 5 min.
      After centrifugation (4 °C, 13,000 x g, 15 min) recover 350 µl of the aqueous phase, add 35 µl NaOAc (pH 5.5), 1 µl GlycoBlue Coprecipitant and 800 µl 100% EtOH and precipitate over night at -20 °C.
      Precipitate the cDNA library by centrifugation (4 °C, 13,000 x g, 15 min), discard the supernatant and wash the pellet with 1 ml 70% EtOH.
      Dissolve the DNA in 20 µl TE buffer, add 3 µl DNA loading buffer and separate the cDNA on a 2.5% agarose gel. The DNA band is excised and isolated using the Wizard SV Gel PCR Clean-Up System.
      Note: Do not denature the DNA, dissolve the gel at 50 °C as shortly as possible!
  12. Submit your cDNA library for high throughput sequencing on an Illumina HiSeq 2000 sequencer performing single-end reads.


    Figure 1. iCLIP procedure of endogenous UPF1. Schematic illustration of the iCLIP procedure. CIP, Alkaline Phosphatase Calf Intestinal. (figure adapted from Reference 1).

Notes

  1. Throughout the entire iCLIP protocol Clear-viewTM Snap-Cap microtubes are used to minimize the absorption of the sample by the plastic and therewith to prevent sample loss.
  2. The usage of the NuPAGE® Novex® 4-12% Bis-Tris Midi Gel in combination with the NuPAGE® MOPS SDS Running Buffer keeps a constant pH 7 during the running procedure and therefore prevents the RNA from alkaline hydrolysis.
  3. To successfully perform iCLIP it is crucial to establish an efficient immunoprecipitation (IP) protocol optimized for your RNA binding protein of interest. Different antibodies and various buffer conditions have to be tested.
  4. For every RNA-binding protein, different RNase I concentrations have to be tested in the limited RNase digestion step in order to obtain RNA fragments with a suitable size (80-200 nt) for cDNA library preparation and subsequent high-throughput sequencing. Especially, working with RNase I, which has no nucleotide specificity, requires careful titration of the RNase I working concentration in order to prevent over-digestion of the RNA. RNase I dilutions from 1: 50 up to 1: 2,000 appeared to be a suitable range for an initial titration experiment. Harsh RNase digestion results in a clear band of the protein-RNA adduct on the autoradiogram after Western blotting. Reducing the RNase concentration leads to a shift of the signal towards higher molecular weight due to the increased heterogeneity in size of the RNA bound to the protein (Figure 2). An 80 nt long RNA contributes approximately 26 kDa to the protein-RNA complex.


    Figure 2. Schematic representation of an autoradiograph of immunopurified and 32P-labelled RNA-protein complexes transferred to nitrocellulose membrane. Immunopurification performed without antibody (no Ab), with cells either depleted of the RNA-binding protein (KD) or not irradiated with UV-C light (no CL) are controls for the specificity of the RNA-protein signal. Increasing dilutions of the RNase I (1:50 to 1:2,000) induce a shift to higher molecular weight of the immunopurified RNA-protein complex. The red arrow marks the electrophoretic mobility of the free RNA-binding protein.

  5. To control for the IP specificity, knocking down the protein of interest or omitting the antibody during the IP procedure should prevent the accumulation of the protein-RNA complex. In the same way, the absence of a protein-RNA complex on the autoradiogram of the western blot is expected when cells are not cross-linked with UV-light. The free radiolabelled RNAs result in a signal running at low molecular weight (Figure 2).

Recipes

Note: All buffers used in the iCLIP protocol have to be sterile, RNase-free and clean of contaminating nucleic acids. To minimize contaminations, it is recommended to have an extra set of reagents reserved for iCLIP experiments. All buffers are prepared with MQ-water and if possible treated with DEPC. Duran bottles, glassware and spatulas are baked at 180 °C for 2 h. Polycarbonate or polystyrene materials (e.g. magnetic stirrers) are soaked in 3% hydrogen peroxide or 2 M NaOH for 10 min and extensively rinsed with DEPC-treated water before use. If a buffer cannot be DEPC treated (e.g. Tris buffers), prepare the buffer in DEPC or MQ-water and filter it with a 0.22 µm filter.

  1. DMEM-/- (per 900 ml)
    12 g DMEM/F12 powder
    2.48 g NaHCO3
    Make up to 900 ml with ddH2O
    Adjust pH to 7.2 with 32% HCl or 10 M NaOH
    Sterilize per filtration with bottle top filter (0.22 µm)
  2. DMEM+/+ (per 500 ml)
    Supply 450 ml DMEM-/- with 50 ml FCS and 5 ml P/S
  3. Phosphate-bufferd saline (PBS)
    137 mM NaCl
    10 mM Na2HPO4
    2.7 mM KCl
    2 mM KH2PO4
    Adjust pH to 7.4 with HCl
  4. Hypotonic gentle lysis buffer (RNase-free)
    10 mM Tris-HCl (pH 7.5)
    10 mM NaCl
    2 mM EDTA
    0.5% (v/v) Triton X-100
    Prior to usage supplement with 1x HaltTM protease inhibitor cocktail
  5. IP-wash buffer (RNase-free)
    50 mM HEPES-NaOH (pH 7.5)
    300 mM KCl
    0.05% (v/v) NP-40
    0.5 mM DTT
    Prior to usage supplement with 1x HaltTM protease inhibitor cocktail
  6. High salt wash buffer (RNase-free)
    50 mM Tris-HCl (pH 7.5)
    1 M NaCl
    1 mM EDTA
    1% (v/v) NP-40
    0.1% (w/v) SDS
    0.5% (w/v) sodium deoxychlate
    Prior to usage supplement with 1x HaltTM protease inhibitor cocktail
  7. PNK buffer (RNase-free)
    50 mM Tris-HCl (pH 7.5)
    10 mM MgCl2
    5 mM DTT
    Prior to usage supplement with 1x HaltTM protease inhibitor cocktail
  8. PNK buffer w/o DTT (RNase-free)
    50 mM Tris-HCl (pH 7.5)
    10 mM MgCl2
    Prior to usage supplement with 1x HaltTM protease inhibitor cocktail
  9. Proteinase K buffer (RNase-free)
    50 mM Tris-HCl (pH 7.5)
    50 mM NaCl
    1% (w/v) SDS
    10 mM EDTA
  10. TE buffer (RNase-free)
    10 mM Tris-HCl (pH 7.5)
    1 mM EDTA
  11. 10x Tris-borate buffer (TBE) (RNase-free)
    890 mM Tris
    890 mM Boric acid
    20 mM EDTA (pH 8)
  12. Acidic phenol
    Melt phenol at 60 °C
    Equilibrate against 2x MQ-water and 0.1% hydroxyquinolone
    Aliquot the phenol-water suspension in 50 ml falcon tubes and stored at -20 °C
  13. Solution A (RNase-free)
    20% Polyacrylamide (29:1)
    1x TBE
    8 M Urea
  14. Solution B (RNase-free)
    1x TBE
    8 M Urea
  15. Loading buffer for PAA-urea gel (RNase-free)
    20 mM EDTA (pH 8.0)
    91% (v/v) deionized formamide
    0.5% (w/v) bromophenol blue
    0.5% (w/v) xylene cyanol
  16. 10x DNA loading buffer
    0.6% (w/v) SDS
    62.5% (v/v) glycerol
    0.5% (w/v) bromophenol blue
  17. DEPC treated water/buffer
    Water/buffer
    0.1% (v/v) DEPC
    Stir over night
    Autoclave twice in order to inactivate DEPC
  18. 3 M NaOAc (pH 4.6 and 5.5) (RNAse-free)
    3 M NaOAc
    Adjust pH to 4.6 and 5.5 with 32% HCl
  19. RNA ligation mix
    28 µl MQ-water
    5 µl 10x reaction buffer for T4 RNase ligase
    5 µl 1 mg/ml BSA 
    1 µl RNasin
    10 µl 20 µM 3’ RNA linker
    1 µl 10 U/µl T4 RNA ligase
  20. Kinase mix
    38 µl MQ-water
    6 µl 10x reaction buffer A (for forward reaction)
    10 µl of 10 µCi/µl γ-P32-ATP
    6 µl 10 U/µl T4 Polynucleotide Kinase
  21. CircLigation mix
    0.8 µl 10x CircLigase buffer II
    0.4 µl 50 mM MnCl2
    0.3 µl CircLigase II
  22. Oligo annealing mix
    21 µl MQ-water
    4 µl 1 mg/ml BSA 
    4 µl Buffer 4
    1 µl 10 µM Cut Oligo
  23. PCR master mix
    2.8 µl 100 µM P5_Solexa
    2.8 µl 100 µM P3_Solexa 
    194.5 µl MQ-water

Acknowledgments

We thank R. Bruggmann and S. Neuenschwander (Institute of Cell Biology, University of Bern, Bern, Switzerland) for initial bioinformatics support, and H. le Hir (Institut de Biologie, École Normale Supérieure, Paris, France) for technical hints with the CLIP. This protocol is optimized to investigate the RNA-binding sites of UPF1 and is based on previously described studies (Konig et al., 2010; Konig et al., 2011; Hafner et al., 2010). Further, we want to thank the Group of Mihaela Zavolan (Swiss Institute of Bioinformatics, Basel, Switzerland), Jernej Ule (Medical Research Council Laboratory of Molecualar Biology, Cambridge, UK) for technical support in setting up these experiments, and Andreas R. Gruber (Swiss Institute of Bioinformatics, Basel, Switzerland) for performing the bioinformatics analysis.

References

  1. Zünd, D., Grüber, A. R., Zavolan, M. and Mühlemann, O. (2013). Translation-dependent displacement of UPF1 from coding sequences causes its enrichment in 3' UTRs. Nat Struct Mol Biol 20(8): 936-943.
  2. König, J., Zarnack, K., Rot, G., Curk, T., Kayikci, M., Zupan, B., Turner, D. J., Luscombe, N. M. and Ule, J. (2010). iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat Struct Mol Biol 17(7): 909-915.
  3. König, J., Zarnack, K., Rot, G., Curk, T., Kayikci, M., Zupan, B., Turner, D. J., Luscombe, N. M. and Ule, J. (2011). iCLIP-transcriptome-wide mapping of protein-RNA interactions with individual nucleotide resolution. JoVE (50).
  4. Hafner, M., Landthaler, M., Burger, L., Khorshid, M., Hausser, J., Berninger, P., Rothballer, A., Ascano, M., Jr., Jungkamp, A. C., Munschauer, M., Ulrich, A., Wardle, G. S., Dewell, S., Zavolan, M. and Tuschl, T. (2010). Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell 141(1): 129-141.

简介

mRNA的命运,特别是其稳定性,定位和翻译速率由装配到信使核糖核蛋白(mRNP)复合物的RNA结合蛋白调节。 为了研究无义介导的mRNA衰变(NMD)的核心因子UPF1的转录组范围的RNA结合位点,我们进行单个核苷酸分辨率的UV交联和免疫沉淀(iCLIP)(Zund等,/em>,2013),然后进行高通量测序。 优化所提出的方案以研究UPF1的RNA结合位点并且基于先前描述的研究(Konig等人,2010; Konig等人,2011; Hafner等人,2010)。 我们要感谢Mihaela Zavolan(瑞士巴塞尔瑞士生物信息学研究所)和Jernej Ule(英国剑桥的分子生物学医学研究委员会实验室)组织这些实验的技术支持。

Fett,C.,DeDiego,M.L.,Regla-Nava,J.A.,Enjuanes,L.and Perlman,S。(2013)。 通过用小鼠免疫在老年小鼠中完全保护免受严重急性呼吸综合征冠状病毒介导的致死性呼吸道疾病 - 适应缺乏E蛋白的病毒。 Virol 87(12):6551-6559。
  • Netland,J.,DeDiego,M.L.,Zhao,J.,Fett,C.,Alvarez,E.,Nieto-Torres,J.L.,Enjuanes,L.and Perlman, 在E蛋白中缺失的减毒性严重急性呼吸综合征冠状病毒的免疫保护免于致命的呼吸道疾病。 a> Virology 399(1):120-128。
  • Zhao,J.,Zhao,J.and Perlman,S。(2010)。 T细胞应答对于严重急性呼吸综合征冠状病毒感染的小鼠中的临床疾病和病毒清除是必需的。 84(18):9318-9325。
  • ...
  • Trypsin-EDTA (T/E) (BioConcept, Amimed, catalog number: 5-5iF00-H)
  • Liquid nitrogen
  • DEPC BioChemica (AppliChem GmbH, catalog number: A0881, 0250)
  • 100x HaltTM Protease Inhibitor Cocktail (Thermo Fisher Scientific, catalog number: 1861279)
  • RNase I (cloned) (100 U/µl) (Life Technologies, Ambion®, catalog number: AM2294)
  • NaCl
  • Turbo DNase(Life Technologies,Ambion ,目录号:AM2238)
  • 山羊抗RENT1抗体(Bethyl Laboratories,目录号:A300-038A)(UPF1也称为RENT1)。
  • 苯酚
  • 氯仿
  • 异戊醇
  • Dynabeads Protein G(Life Technologies,目录号:10004D)
  • 碱性磷酸酶小牛肠(CIP)(New England Biolabs,目录号:M0290S)
  • T4 RNA连接酶(10U /μl)(Thermo Fisher Scientific,目录号:EL0021)
  • RNasin(Dundee Cell Products,目录号:RS3100)
  • 3'RNA接头(5'-P-UGAGAUCGGAAGAGCGGUUCAG-嘌呤霉素-3')(Microsynth AG; http://www.microsynth .ch/
    注意:RNA以5nmol等分试样递送,储存在-80℃,在使用前溶于25μlMQ-水中。
  • γ-P32-ATP(10μCi/μl)(HARTMANN ANALYTIC GmbH,目录号:SCP-401)
  • T4多核苷酸激酶(10U /μl)(Thermo Fisher Scientific,目录号:EK0031)
  • 4x NuPAGE LDS样品缓冲液(Life Technologies,目录号:NP0007)
  • 20x NuPAGE MOPS SDS运行缓冲液(Life Technologies,目录号:NP0001)
  • Prestained Protein Ladder(宽范围)(New England Biolabs,目录号:P7710S)
  • GlycoBlue 共沉淀剂(15mg/ml)(Life Technologies,目录号:AM9515)
  • 蛋白酶K(重组PCR级)(Roche Diagnostics,目录号:03 115 887 001)
  • RT-引物(Microsynth AG)
    寡核苷酸序列: 5'-P-NNNNAACAGATCGGAAGAGCGTCGTGGATCCTGAACCGC-3'
  • dNTP混合物(各10mM)(Thermo Fisher Scientific,目录号:R0192)
  • AffinityScript TM Multi-Temp RT(Agilent,目录号:600109-51)
  • 40%丙烯酰胺溶液(mix 19:1,分子生物学级)(AppliChem GmbH,目录号:A3658)
  • 四甲基乙二胺(TEMED)(Sigma-Aldrich,目录号:T22500-100ML)
  • 过硫酸铵(APS)(Sigma-Aldrich,目录号:A3678-100G)
  • PlusOne Repel-Silane ES(GE Healthcare,目录号:17-1332-01)
  • 低分子量DNA梯(新英格兰Biolabs,目录号:N3233L)
  • SYBR 金核酸凝胶染料(Life Technologies,目录号:S11494)
  • CircLigase TM II ssDNA连接酶(震中,目录号:CL9025K)
  • Cut-Oligo(5'-GTTCAGGATCCACGACGCTCTTCAAAA-3')(Microsynth AG)
  • BamHI(New England Biolabs,目录号:R0136S)
  • P5_Solexa引物(Microsynth AG)
    寡核苷酸序列: 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3'
  • P3_Solexa引物(Microsynth AG)
    寡核苷酸序列: 5'-CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT-3'
  • Maximo Taq DNA 2X-Mastermix(GeneOn,目录号:S114)
  • DMEM -/- (每900ml)(参见配方)
  • DMEM +/+(每500ml)(参见配方)
  • 磷酸盐缓冲盐水(PBS)(pH 7.4)(见Recipes)
  • 低渗温和裂解缓冲液(pH 7.5)(无RNase)(见配方)
  • IP洗涤缓冲液(不含RNase)(参见配方)
  • 高盐洗涤缓冲液(pH 7.5)(无RNase)(见配方)
  • PNK缓冲液(pH 7.5)(不含RNase)(见配方)
  • PNK缓冲液,无DTT(pH 7.5)(无RNase)(见配方)
  • 蛋白酶K缓冲液(pH 7.5)(无RNase)(见配方)
  • TE缓冲液(pH 7.5)(不含RNase)(参见配方)
  • 10x Tris-硼酸盐缓冲液(TBE)(pH 8.0)(不含RNase)(参见配方)
  • 酸性苯酚(参见配方)
  • 解决方案A(不含RNase)(参见配方)
  • 溶液B(不含RNase)(参见配方)
  • PAA-尿素凝胶(pH 8.0)(不含RNase)的装载缓冲液(参见配方)
  • 10x DNA上样缓冲液(见配方)
  • DEPC处理的水/缓冲液(见配方)
  • 3 M NaOAc(pH 4.6和5.5)(无RNA酶)(参见配方)
  • RNA连接混合物(参见配方)
  • 激酶混合(参见配方)
  • CircLigation mix(见配方)
  • 低聚退火混合物(参见配方)
  • PCR主混合物(参见配方)
  • 设备

    1. 纯水系统:PURELAB Priama(Prima 7)和PURELAB ULTRA (Ultra Genetic)(ELGA LabWater)
      注意:在此协议中,来自ELGA系统的纯水称为MQ-水。
    2. CO 2培养箱(BINDER GmbH,型号:9140-0047)
    3. Clear-view TM Snap-Cap微管(1.5ml,天然,低保留)(Sigma-Aldrich,目录号:T4816-250EA)
    4. Multiply -Pro 0.2ml Biosphere (Sarstedt AG,型号:72.727)
    5. 过滤嘴(10μl,20μl和200μl)(Axon Lab AG,目录号:AL60X10,AL60X20,AL60X200)
    6. 过滤嘴(1,250μl)(Greiner Bio-One GmbH,目录号:7.750.261)
    7. 15cm组织培养皿(TPP Techno Plastic Products,目录号:93150)
    8. Stratalinker 2400(254nm)(Stratagene ,目录号:400076)
    9. PCR管
    10. 电池升降器(TPP Techno Plastic Products,型号:99010)
    11. Saran换装
    12. 苏格兰
    13. GP Millipore Express PLUS膜(0.22μm)(500ml漏斗,45mm颈部尺寸)(EMD Millipore,目录号:SCGPT05RE)
    14. DynaMag TM -2磁体(Life Technologies,目录号:12321D)
    15. 水浴(温度可调)
    16. Eppendorf离心机5415R,带转子F45-24-11(Eppendorf,型号:022621459和022636502)
    17. Criterion TM细胞(Bio-Rad Laboratories,目录号:165-6001)
    18. NuPAGE Novex 4-12%Bis-Tris Midi Gel(1.0mmx,12 + 2孔)(Life Technologies,目录号:WG1401BOX)
    19. Midi Gel Adaptor(Life Technologies,型号:WA0999)
    20. iBlot ®凝胶转移装置(EU)(Life Technologies,型号:IB1001EU)
    21. iBlot Transfer Stack(硝基纤维素,常规尺寸)(Life Technologies,目录号:IB3010-01)
    22. 10ml血清移液器
    23. POWER PAC 3000(Bio-Rad Laboratories)
    24. Lab循环仪梯度,配备有Thermoblock 96(SensoQuest GmbH,型号:011-101和012-103)
    25. 印花纸(Genentech,目录号:BP 002 46579)
    26. FUJI屏幕
    27. FUJIFILM FLA-3000磷光仪
    28. Costar Spin-X 离心柱(Corning,型号:8161)
    29. 用于具有1mm厚隔离物和梳子(自制)的180×120mm凝胶的垂直PAA凝胶运行装置
    30. 回形针(51毫米)(Lyreco,目录号:161.791)
    31. 铝板(90×175mm,厚度3mm)
    32. UV台(365 nm)(自制)
    33. 1ml注射器(BD,Plastipak TM,目录号:300013)

    程序

    1. 细胞培养和UV交联
      1. Hela细胞在补充有100U/ml青霉素,100μg/ml链霉素和10%胎牛血清的10个15cm直径培养皿中的Dulbecco改良的Eagle培养基(DMEM)中培养直到它们达到80%汇合。
      2. 弃去培养基,用12ml冰冷的PBS缓冲液覆盖细胞
      3. 使细胞经受150mJ/cm 2 UV-C光。 在照射期间,将盘放置在冰上
      4. 照射后,用10ml新鲜的冰冷PBS覆盖细胞,并使用细胞提升器刮下
      5. 通过离心(4℃,200xg,5分钟)将细胞沉淀在两个50ml的falcon管中,在液氮中骤冷并储存直至在-80℃下使用。
    2. 有限的RNase I消化和UPF1免疫沉淀
      1. 将细胞在冰上解冻并在7ml低渗温和裂解缓冲液中裂解20分钟。 通过离心(4℃,13,000xg,15分钟)除去细胞碎片。 回收上清液 通过加入350μl的3M NaCl将NaCl浓度调节至150mM
      2. 对于部分RNA酶消化,将RNase I在低渗温和裂解缓冲液中以1:1000稀释,并且用70μlRNase I稀释液和35μlTurbo DNase在37℃水浴中轻微摇动3分钟处理细胞裂解物。
        注意:对于每个进行iCLIP的RNA结合蛋白,必须重新调整RNase I消化步骤,以获得与感兴趣的蛋白质结合的80-200nt长的RNA片段,适用于cDNA文库制备和随后的高(见注释)。
      3. 将RNase I处理的裂解物在冰上放置3分钟
      4. 加入28μl山羊抗RENT1抗体,并在4℃下尾部旋转1小时
      5. 将240μlDynabeads G蛋白G用1ml PBS缓冲液洗涤两次,并用1ml低渗温和裂解缓冲液洗涤一次。为了收集磁性Dynabeads ,使用DynaMag TM -2磁性机架。
      6. 在用UPF1/RENT1抗体预孵育裂解物1小时后,加入洗涤过的珠,并在4℃下再孵育1小时。
      7. 取出细胞裂解液,用IP洗涤缓冲液洗涤珠子3次
    3. RNA的去磷酸化,随后3'-RNA-接头的连接
      1. 为了使与UPF1共价结合的RNA片段去磷酸化,将Dynabeads重悬于60μl脱磷酸化混合物[55.25μlMQ-水,6.5μl10×NEBuffer3,3.25μl碱性磷酸酶小牛肠(CIP)]中并在37℃下温育20分钟。
      2. 将珠用1ml高盐洗涤缓冲液洗涤两次用于严格洗涤,并用1ml PNK缓冲液洗涤两次
      3. 在最后一次洗涤步骤后,小心地除去上清液,将珠子在50μlRNA连接混合物中温育。 3'RNA接头的过夜连接在0.2ml PCR管中进行 置于16℃的PCR仪中。盖子保持在恒定的50℃,以防止样品冷凝
    4. RNA的放射性标记和RNA-UPF1复合物的分离
      1. 样品用1ml PNK缓冲液洗涤两次,并通过与60μl激酶混合物在37℃下温育,在800rpm下振荡45分钟,对RNA进行放射性标记。
      2. 将结合到珠上的放射性标记的样品用不含DTT的PNK缓冲液洗涤四次(在第二次洗涤步骤之后保持放射性缓冲液,并用于步骤4i中的热定位标记)。
      3. 通过在45μl1x NuPAGE LDS样品缓冲液中在90℃下烹饪5分钟,从珠粒上洗脱蛋白质-RNA复合物。
      4. 凝胶流动装置使用BioRAD标准室,中间凝胶接头和预制的NuPAGE Novex 4-12%Bis-Tris midi凝胶组装。流动室装有500ml 1x NuPAGE MOPS SDS运行缓冲液
      5. 将洗脱的蛋白质-RNA样品(40μl)加载到NuPAGE Novex凝胶上,紧邻6μl预染色的蛋白梯,并在200V下分离45-60分钟,直到溴酚蓝运行前为约离开凝胶。
      6. 拆卸凝胶装置并切断由溴酚蓝色带标记的正面。凝胶的下部含有未掺入的γ-P32-ATP,并作为固体放射性废物处理。
      7. 使用iBlot系统将蛋白质-RNA加合物转移到硝酸纤维素膜上(有关详细说明,请参阅制造商提供的教程)。
      8. 在7分钟的转移过程之后,转移堆叠被拆卸并作为固体放射性废物处理
      9. 将硝酸纤维素膜包裹在纱布包装中,并且在膜的边缘处附着三个定位标记[1μl在步骤4b中放在一边的热PNK缓冲液铺在小的吸干纸条(1.5×8mm)上并固定在用苏格兰一小块苏珊包装。这些标记对于在步骤4k中适当地对准放射自显影以从UPF1-mRNA复合物与硝酸纤维素膜分离是重要的。]
      10. 为了检测硝酸纤维素膜上的P32标记的RNA-UPF1复合物,将FUJI屏幕暴露1小时,并在FUJIFILM FLA-3000磷光仪上进行分析。
      11. 膜的放射自显影图以真实抓取格式印刷并在定位标记的帮助下与硝酸纤维素膜对准。 切下含有RNA-UPF1复合物的硝酸纤维素膜切片,并使用1,250μl移液管吸头将其研磨成小块。
      12. 通过将膜片与200μl添加有20%(v/v)蛋白酶K的蛋白酶K缓冲液在50℃下温育1小时来分离RNA。
      13. 重复洗脱以最大化洗脱效率
      14. 使用costar Spin-X超声离心柱过滤(以2000×g离心2分钟)合并的洗脱级分(终体积400μl)。
    5. 苯酚/氯仿提取和RNA沉淀
      1. 向过滤的RNA样品中加入一体积的酸性酚:氯仿:异戊醇(25:24:1),并以1,000rpm(不涡旋)摇动5分钟。
      2. 旋转样品(16,000×g,4℃,10分钟),并将水相(350μl)转移到新管中。
      3. 加入35μl3M NaOAc(pH4.6),2μlGlycoBlue共沉淀剂,400μl异丙醇; 混合并在-20℃下沉淀RNA过夜
      4. 通过离心(16,000×g,4℃,15分钟)沉淀RNA,用1ml 70%EtOH洗涤并溶解于5.5μlMQ-水中。
    6. 逆转录
      1. 将样品转移到0.2 ml PCR管中
      2. 向RNA中加入0.5μlRNasin,0.5μldNTP(各10mM)和0.5μlRT-引物(0.5pmol /μl)并加热至70℃5分钟(在PCR热循环仪中)。 >
      3. 在将样品缓慢冷却至25℃(温度梯度:1.8℃/min)后,加入3μl以下RT混合物(1μl10x AffinityScript RT缓冲液,1μl0.1M DTT,1μlAffinity Script Multi-Temp RT) 已添加。
      4. 使用以下热程序继续逆转录:
        25°C   
        15分钟
        50°C
        60分钟
        4°C   
        保持
      5. 将cDNA转移到1.5ml试管中,通过加入90μlTE缓冲液,0.5μlGlycoBlue共沉淀剂,10μlNaOAc(pH 5.5),250μl100%EtOH并随后在-20℃下温育过夜沉淀。 br />
      6. 通过离心(4℃,16,000×g,15分钟)沉淀cDNA。
      7. 用70%EtOH洗涤沉淀
      8. 将cDNA重悬于10μlPAA - 尿素凝胶上样缓冲液中,并在70℃下煮5分钟。
      9. 将2μl低分子量标记物与2μl6x上样缓冲液和8μlPAA - 尿素凝胶上样缓冲液混合(不要煮标记物)。
    7. 铸造6%TBE-脲凝胶
      1. 小心清洗并干燥玻璃板(180 x 120 mm)。
      2. 用柔软的纸巾将PlusOne反光硅烷ES涂在玻璃板的内表面上
      3. 组装玻璃板和间隔(1 mm厚度),并用两个文件夹固定设置,两个文件夹位于间隔垫片的中心。 将组装的玻璃板放在空的提箱上。 将凝胶浇铸在水平位置使得填料的密封变得多余。
      4. 通过混合7.5ml溶液A与17.5μl溶液B制备25ml 6%凝胶溶液。通过加入125μl10%APS和12.5μlTEMED开始聚合。
      5. 将凝胶溶液用10ml血清移液管施加到玻璃板的将插入梳的一侧的边缘。 玻璃板上的表面张力和轻微敲击确保了凝胶溶液的均匀和无气泡分布
      6. 引入梳子,用附加的文件夹固定玻璃板。
      7. 聚合凝胶1小时。
    8. 运行6%TBE-尿素凝胶
      1. 将用铝板组装的凝胶夹入凝胶流动室(90×175×3mm铝板确保均匀的热分布,并且使凝胶中的样品均匀流动),并用1×TBE缓冲液填充该装置。 />
      2. 用注射器小心地清洗插槽。
      3. 预运行凝胶(10 W,30分钟)。
      4. 再次洗涤插槽。
      5. 加载低分子量DNA梯和cDNA样品,在其间留下2-3个空泳道
      6. 运行凝胶(5 W,70分钟),直到溴酚蓝色条带在凝胶底部以上2厘米。
    9. 分离cDNA
      1. 切割凝胶,以便从包含样品的泳道分离含有梯子的泳道
      2. 在1x TBE缓冲液中用1x SYBR金染色含标记物的凝胶部分(15分钟),并将含有样品的凝胶部分包裹在saran包装中。
      3. 可视化365nm紫外线桌上的DNA梯形图,并在透明度上复制尺寸梯形图的位置。
      4. 借助于显示尺寸梯子(放置在未染色的凝胶部分旁边)的透明度,切除含有覆盖150-300nm大小范围的cDNA的凝胶部分。
      5. 用注射器柱塞(从1ml注射器)压碎凝胶块,并用400μlTE缓冲液(在2,000rpm,37℃,2小时摇动)提取cDNA。
      6. 用Costar Spin-X 离心柱(2分钟,2,000×g)过滤上清液。
      7. 通过加入1μlGlycoBlue共沉淀,40μlNaOAc(pH 5.5)和1ml 100%EtOH沉淀cDNA,并在-20℃下孵育1小时。
      8. 通过离心(4℃,16,000×g,15分钟)沉淀cDNA。
      9. 用1ml 70%EtOH洗涤沉淀
      10. 将cDNA溶解在6.7μlMQ-水中
    10. 通过环化和随后的线性化将5'引物引入cDNA
      1. 为了进行环化,将1.5μlCircLigation混合物加入到6.7μlcDNA中,然后在PCR热循环仪中在60℃温育1小时。
      2. 为了线性化单链环状cDNA,将DNA剪切寡核苷酸退火到RT引物中产生含有BamHI位点的双链DNA延伸的区域。
        准备寡核苷酸退火混合和添加30微升到环状cDNA的。 将样品加热至90℃并缓慢冷却至25℃(在PCR热循环仪中,1℃/min温度梯度)。
      3. 为了切割环状cDNA,加入2μlBamHI限制酶并在37℃下孵育45分钟
      4. 通过加入50μlTE缓冲液,0.5μlGlycoBlue共沉淀,10μlNaOAc(pH 5.5)和250μl100%乙醇沉淀线性化的cDNA,然后在-20℃孵育过夜。
      5. 通过离心(4℃,13,000×g,15分钟)沉淀线性化的cDNA。
      6. 用70%EtOH洗涤沉淀
      7. 将沉淀的cDNA溶于50μlMQ-水中
    11. PCR扩增cDNA
      使用P5_Solexa和P3_Solexa引物通过PCR引入高通量Illumina测序所需的衔接头。
      1. 引导PCR
        为了确定cDNA文库的适当扩增所需的循环数,进行100μl测试PCR。为了设置测试PCR,将10μlcDNA,40μlPCR主混合物和50μlMaximo Taq DNA mastermix组合,并运行以下PCR程序:
        95℃5分钟
        34个循环(95℃45秒,50℃80秒,72℃1分钟) 为了在循环20,22,24,26,28,30,32和34后分析中间PCR产物,取12μl样品并与3μl10x DNA上样缓冲液混合。
        每个样品在2.5%琼脂糖凝胶上分开7.5μl以确定产生刚好足够的PCR产物所需的循环数(N <阈值),导致在琼脂糖凝胶上可见的条带。应避免过度扩增,因为它大大降低了测序文库的复杂性
      2. 制备PCR
        对于cDNA文库的制备性PCR扩增,以下列循环进行四个100μlPCR反应:
        95℃5分钟
        (95℃持续45秒,50℃持续80秒,72℃持续1分钟)的N个阈值循环。
        72℃5分钟
      3. 清理制备PCR产品
        将四种PCR产物合并,与400μl苯酚:氯仿:异戊醇(25:24:1)混合,并在室温下尾部旋转5分钟。
        离心(4℃,13,000xg,15分钟)后,回收350μl水相,加入35μlNaOAc(pH 5.5),1μlGlycoBlue共沉淀剂和800μl100%EtOH,并沉淀在夜间在-20°C。
        通过离心(4℃,13,000xg,15分钟)沉淀cDNA文库,弃去上清液并用1ml 70%EtOH洗涤沉淀。
        将DNA溶解在20μlTE缓冲液中,加入3μlDNA加样缓冲液,并在2.5%琼脂糖凝胶上分离cDNA。使用Wizard SV凝胶PCR清除系统切除并分离DNA条带 注意:不要使DNA变性,应尽快在50°C溶解凝胶!
    12. 提交您的cDNA文库,在Illumina HiSeq 2000测序仪上进行高通量测序,进行单端读数

      图1.内源性UPF1的iCLIP程序。 iCLIP程序的示意图。 CIP,碱性磷酸酶小牛肠道。 (从参考文献1改编的图)

    笔记

    1. 在整个iCLIP协议中,Clear-view TM Snap-Cap微管用于最小化塑料对样品的吸收,从而防止样品损失。
    2. NuPAGE Novex 4-12%Bis-Tris Midi Gel与NuPAGE MOPS SDS运行缓冲液的组合使用保持 在运行程序期间保持恒定的pH 7,因此防止RNA进行碱性水解。
    3. 要成功地执行iCLIP,关键是建立一个有效的免疫沉淀(IP)协议优化您感兴趣的RNA结合蛋白。 必须测试不同的抗体和各种缓冲液条件。
    4. 对于每种RNA结合蛋白,必须在有限的RNA酶消化步骤中测试不同的RNA酶I浓度,以获得具有用于cDNA文库制备和随后的高通量测序的合适大小(80-200nt)的RNA片段。特别是,与没有核苷酸特异性的RNase I一起,需要仔细滴定RNase I的工作浓度以防止RNA的过度消化。 1:50至1:2,000的RNA酶I稀释度似乎是初始滴定实验的合适范围。 Harsh RNA酶消化在蛋白质印迹后在放射自显影图上产生蛋白质-RNA加合物的清晰带。降低RNase浓度导致信号向更高分子量的转移,这是由于与蛋白质结合的RNA的大小的异质性增加(图2)。 80nt的RNA对蛋白质-RNA复合物贡献大约26kDa。


      图2.免疫纯化和<32> P标记的RNA-蛋白复合物转移到硝酸纤维素膜的放射自显影图的示意图。在无抗体(无Ab)的情况下进行免疫纯化,缺乏RNA结合蛋白(KD)或未用UV-C光照射(无CL)是RNA-蛋白信号特异性的对照。 RNase I(1:50至1:2,000)的增加的稀释物诱导转移至较高分子量的免疫纯化的RNA-蛋白复合物。红色箭头标记游离RNA结合蛋白的电泳迁移率。

    5. 为了控制IP特异性,在IP过程中敲低目标蛋白质或省略抗体应当防止蛋白质-RNA复合物的积累。以相同的方式,当细胞不与UV光交联时,预期在蛋白质印迹的放射自显影图上不存在蛋白质-RNA复合物。游离放射性标记的RNA导致信号以低分子量运行(图2)。

    食谱

    注意:iCLIP协议中使用的所有缓冲液必须是无菌的,无核糖核酸酶的并且清洁污染的核酸。为了最小化污染,建议为iCLIP实验保留一套额外的试剂。所有缓冲液用MQ-水制备,并且如果可能用DEPC处理。 Duran瓶,玻璃器皿和铲子在180℃烘烤2小时。将聚碳酸酯或聚苯乙烯材料(例如磁力搅拌器)在3%过氧化氢或2M NaOH中浸泡10分钟,并在使用前用DEPC处理的水充分冲洗。如果缓冲液不能被DEPC处理(例如Tris缓冲液),则在DEPC或MQ-水中制备缓冲液,并用0.22μm过滤器过滤。

    1. DMEM -/- (每900ml) 12克DMEM/F12粉末
      2.48g NaHCO 3水溶液 用ddH 2 O·
      补充至900ml 用32%HCl或10M NaOH将pH调节至7.2 用瓶顶过滤器(0.22μm)过滤灭菌
    2. DMEM +/+(每500ml) 供应450毫升DMEM -/- 与50毫升FCS和5毫升P/S
    3. 磷酸盐缓冲盐水(PBS)
      137 mM NaCl 10mM Na 2 HPO 4
      2.7 mM KCl
      2mM KH 2 PO 4 sub/
      用HCl
      调节pH至7.4
    4. 低渗温和裂解缓冲液(不含RNase)
      10mM Tris-HCl(pH7.5) 10mM NaCl 2mM EDTA 0.5%(v/v)Triton X-100 在使用之前补充1x Halt TM 蛋白酶抑制剂混合物
    5. IP洗涤缓冲液(不含RNase)
      50mM HEPES-NaOH(pH7.5) 300 mM KCl
      0.05%(v/v)NP-40
      0.5 mM DTT
      在使用之前补充1x Halt TM 蛋白酶抑制剂混合物
    6. 高盐洗涤缓冲液(不含RNase)
      50mM Tris-HCl(pH7.5) 1 M NaCl
      1mM EDTA
      1%(v/v)NP-40
      0.1%(w/v)SDS
      0.5%(w/v)脱氧胆酸钠 在使用之前补充1x Halt TM 蛋白酶抑制剂混合物
    7. PNK缓冲液(不含RNase)
      50mM Tris-HCl(pH7.5) 10mM MgCl 2/
      5 mM DTT
      在使用之前补充1x Halt TM 蛋白酶抑制剂混合物
    8. 无DTT(无RNase)的PNK缓冲液
      50mM Tris-HCl(pH7.5) 10mM MgCl 2/
      在使用之前补充1x Halt TM 蛋白酶抑制剂混合物
    9. 蛋白酶K缓冲液(不含RNase)
      50mM Tris-HCl(pH7.5) 50mM NaCl 1%(w/v)SDS
      10 mM EDTA
    10. TE缓冲液(无RNase)
      10mM Tris-HCl(pH7.5) 1mM EDTA
    11. 10x Tris-硼酸盐缓冲液(TBE)(无RNase)
      890mM Tris 890mM硼酸 20mM EDTA(pH8)
    12. 酸性酚
      在60℃下熔融苯酚
      平衡2×MQ-水和0.1%羟基喹啉
      将酚 - 水悬浮液在50ml falcon管中等分,并储存在-20℃下
    13. 解决方案A(无RNase)
      20%聚丙烯酰胺(29:1) 1x TBE
      8 M尿素
    14. 溶液B(无RNase)
      1x TBE
      8 M尿素
    15. PAA-尿素凝胶的装载缓冲液(不含RNase)
      20mM EDTA(pH8.0) 91%(v/v)去离子甲酰胺 0.5%(w/v)溴酚蓝
      0.5%(w/v)二甲苯蓝
    16. 10x DNA上样缓冲液
      0.6%(w/v)SDS
      62.5%(v/v)甘油 0.5%(w/v)溴酚蓝
    17. DEPC处理的水/缓冲液
      水/缓冲液
      0.1%(v/v)DEPC
      搅拌过夜
      高压灭菌两次以停用DEPC
    18. 3 M NaOAc(pH 4.6和5.5)(无RNA酶)
      3 M NaOAc
      用32%HCl调节pH至4.6和5.5
    19. RNA连接混合物
      28μlMQ-水
      5μl用于T4核糖核酸酶连接酶的10x反应缓冲液
      5μl1mg/ml BSA
      1微升RNasin
      10μl20μM3'RNA接头
      1μl10 U /μlT4 RNA连接酶
    20. 激酶混合物
      38μlMQ-水
      6μl10x反应缓冲液A(用于正向反应)
      10μl10μCi/μlγ-P32-ATP
      6μl10 U /μlT4多核苷酸激酶
    21. CircLigation mix
      0.8μl10×CircLigase缓冲液II 0.4μl50mM MnCl 2/v/v 0.3μlCircLigase II
    22. 低温退火混合物
      21μlMQ-水
      4μl1mg/ml BSA
      4μl缓冲液4
      1μl10μM切割寡核苷酸
    23. PCR主混合物
      2.8μl100μMP5_Solexa
      2.8μl100μMP3_Solexa 
      194.5μlMQ-水

    致谢

    我们感谢用于初始生物信息学支持的R. Bruggmann和S. Neuenschwander(伯尔尼大学的细胞生物学研究所),以及H.le Hir(Institut de Biologie,ÉcoleNormaleSupérieure,Paris,France),用于技术提示剪辑。该方案被优化以研究UPF1的RNA结合位点,并且基于先前描述的研究(Konig等人,2010; Konig等人,2011; Hafner et al。, 2010)。此外,我们要感谢Mihaela Zavolan(瑞士巴塞尔瑞士生物信息学研究所),Jernej Ule(英国剑桥的分子生物学医学研究委员会实验室)在设置这些实验方面的技术支持,Andreas R. Gruber(Swiss Institute of Bioinformatics,Basel,Switzerland)进行生物信息学分析。

    参考文献

    1. Zünd,D.,Grüber,A.R.,Za​​volan,M。和Mühlemann,O。(2013)。 翻译依赖于UPF1从编码序列的置换导致其在3'UTR的富集。 Nat Struct Mol Biol 20(8):936-943。
    2. König,J.,Zarnack,K.,Rot,G.,Curk,T.,Kayikci,M.,Zupan,B.,Turner,D.J.,Luscombe,N.M.and Ule,J.(2010)。 iCLIP揭示了hnRNP颗粒在单个核苷酸分辨率剪接中的功能。 Nat Struct Mol Biol 17(7):909-915。
    3. König,J.,Zarnack,K.,Rot,G.,Curk,T.,Kayikci,M.,Zupan,B.,Turner,D.J.,Luscombe,N.M.and Ule,J。 iCLIP-转录组范围内的蛋白质-RNA相互作用与单个核苷酸分辨率的映射。 a> JoVE (50)。
    4. Hafner,M.,Landthaler,M.,Burger,L.,Khorshid,M.,Hausser,J.,Berninger,P.,Rothballer,A.,Ascano,M.,Jr.,Jungkamp,AC,Munschauer,M Ulrich,A.,Wardle,GS,Dewell,S.,Zavolan,M。和Tuschl,T。(2010)。 PAR-CLIP对RNA结合蛋白和微小RNA靶位点的全转录组鉴定。 141(1):129-141。
    • 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: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
    引用:Zünd, D. and Mühlemann, O. (2014). Individual-nucleotide-resolution UV Cross-linking and Immunoprecipitation (iCLIP) of UPF1. Bio-protocol 4(7): e1085. DOI: 10.21769/BioProtoc.1085.
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