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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）

David Zünd David Zünd
Oliver Mühlemann Oliver Mühlemann

发布: 2014年04月05日第4卷第7期 DOI: 10.21769/BioProtoc.1085 浏览次数: 15245

评审: Anonymous reviewer(s)

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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

English
中文翻译

文章信息

版权信息

© 2014 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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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分类

生物化学 > RNA > RNA-蛋白质相互作用

分子生物学 > RNA > mRNA 转译

生物化学 > 蛋白质 > 免疫检测 > 免疫沉淀（IP）

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