Subject Area:	Biochemistry, Genetics and Molecular Biology
More specific subject area:	Protein-RNA Interactions
Protocol name:	Quick-irCLIP – Rapid infrared adaptor based individual nucleotide resolution UV cross-linking and immunoprecipitation
Reagents/tools:	•5’ DNA Adenylation Kit (NEB, Cat#:E2610L) •QIAquick Nucleotide Removal Kit (Qiagen, Cat#: 28304) •1x Phosphate buffered saline (Fisher Scientific, Cat#: AAJ61196AP) •Tris-HCL, pH 6.5(Fisher Scientific, Cat#: AAJ61787AP) •Tris-HCL, pH 7.4(Fisher Scientific, Cat#: AAJ62778AP) •Tris-HCL, pH 7.8(Fisher Scientific, Cat#: AAJ61944AP) •NaCl (Fisher Scientific, Cat#: S271-500) •Igepal CA-630 (Sigma Aldrich, Cat#: I8896 −50 ML) •SDS (Fisher Scientific, Cat#: BP166-100) •Soidum deoxycholate (Fisher Scientific, Cat#: PI89905) •Urea (Fisher Scientific, Cat#: U15-500) •EDTA pH 8.0 (Fisher Scientific, Cat#: BP118-500) •MgCl2 (Fisher Scientific, Cat#: AA12315A1) •Tween 20 (Fisher Scientific, Cat#: BP337-100) •Dithiothreitol (Fisher Scientific, Cat#: BP172-5) •Polyethylene glycol 400 (Fisher Scientific, Cat#: AAB2199230) •LDS-4x sample buffer (Fisher Scientific, Cat#: NP0007) •Methanol (Fisher Scientific, Cat#: A412-500) •20x MOPS-SDS running buffer (Fisher Scientific, Cat#: NP0001) •20x Transfer buffer (Fisher Scientific, Cat#: NP0006) •Neutral phenol:chloroform (Fisher Scientific, Cat#: PI17908) •Sodium acetate (3 M), pH 5.5 (Fisher Scientific, Cat#: AM9740) •Ethanol (Fisher Scientific, Cat#: 07-678-003) •TBE buffer (Bio-Rad, Cat#: 161-0733) •Magnetic protein G/A beads (Bio-Rad, Cat#: 1614023) •Anti-hnRNP-C (positive control) (Santa Cruz, Cat#: sc-32308) •Protease inhibitor cocktail (Fisher Scientific, Cat#: 78429) •RNase I (Fisher Scientific, Cat#: FEREN0601) •Turbo DNase (Fisher Scientific, Cat#: AM2238) •T4 PNK (NEB, Cat#: M0201S) •RNaseOUT Ribonuclease Inhibitor (Fisher Scientific, Cat#: 10777019) •T4 RNA ligase I (NEB, Cat#: M0204S) •Near infrared protein marker (Bio-Rad, Cat#: 1610374) •Antioxidant (Fisher Scientific, Cat#: NP0005) •Reducing agent (Fisher Scientific, Cat#: NP0004) •Proteinase K (Qiagen, Cat#: 19131) •Glycogen, RNA grade (Fisher Scientific, Cat#: FERR0551) •50bp DNA marker (NEB, Cat#: N3236S) •SYBR safe (Thermofisher Scientific, Cat#: S33012) •SMARTer® smRNA-seq kit for Illumina (Takara, Cat#: 635029) •4–12% protein denaturing precast gels (Fisher Scientific, Cat#: NP0335BOX) •6% TBE precast gels (Fisher Scientific, Cat#: EC6865BOX) •Irdye-800CW-DBCO (LiCor, Cat#: 929-50000) •QIAquick Nucleotide Removal Kit (Qiagen, Cat#: 28304) •Cell scrapers (Fisher Scientific, Cat#: 08-771-1A) •2 mL microcentrifuge tubes (Fisher Scientific, Cat#: 05-402-95) •1.5 mL microcentrifuge tubes (Fisher Scientific, Cat#: 05-402-94) •Low-binding 1.5 mL microcentrifuge tubes (Fisher Scientific, Cat#: 13-698-794) •0.2 mL PCR tubes (Eppendorf, Cat#: 951010006) •Steriflip filters (Fisher Scientific, Cat#: SCGP00525) •Protran 0.45 nitrocellulose membrane (Fisher Scientific, Cat#: 45-004-01 •Western blotting filter paper (Fisher Scientific, Cat#: PI88600) •Razor blades (Fisher Scientific, Cat#: 12-640) •30 gauge needles (Sigma Aldrich, Cat#: Z192341) •Phase-lock heavy columns (VWR, Cat#: 10847-802) •Proteus clarification columns (Fisher Scientific, Cat#: 50-107-8783) •Magnetic microcentrifuge tube rack (Bio-Rad, Cat#: 161-4916) •IP-grade Antibody targeting RBP of interest •/5Phos/CAAGCAGAAGACGGCATACGAAAAAAAAAAAA/iAzideN/AAAAAAAAAAAA oligonucleotide (synthesized at the 1 μmol scale (with an approximate yield of 20 nmol)) •Nanodrop (Fisher Scientific, Cat#: 13-400-519) •Thermal cycler (Agilent, Cat#: G8800A) •Thermomixer (Fisher Scientific, Cat#: 13687717) •Microcentrifuge (Fisher Scientific, Cat#: 05-401-203) •Acetate printing film (Office Depot, Cat#: 542290) •Printer (Office Depot, Cat#: 872049) •Near infrared imager (Bio-Rad, Cat#: 12003154) •UV crosslinker (Fisher Scientific, Cat#: 13-245-221) •Gel and transfer apparatus (Fisher Scientific, Cat#: EI0002)
Experimental design:	Proteins and RNAs in the cells of interest are cross-linked through UV irradiation. The protein of interest is immunoprecipitated along with its cross-linked RNAs. An infrared RNA adaptor allows for visualization and isolation of protein-RNA complexes following Western blotting. Then the protein is digested, and the RNA is purified and used to create a sequencing library. During library preparation, frequent failure of the reverse transcriptase to read through the cross-link site, allows for the resolution of RNA regions involved in protein-RNA interactions at the nucleotide level.
Trial registration:	n/a
Ethics:	n/a