GSM4458899: HEK293T par-clip from HDLBP_2; Homo sapiens; OTHER
Cells were UV crosslinked (365 nm, 0.15 J/cm²), harvested, snap frozen and stored at -80°C. Cell pellets were incubated with 3 cell pellet volumes of lysis buffer (50 mM Tris-HCl [pH 7.4], 100 mM NaCl, 1 % IGEPAL CA-630, 0,1 % SDS, 0,5% sodium deoxycholate, complete EDTA-free protease inhibitor cocktail [Roche]) for 30 min on ice. Cell lysates were cleared (13.000 rpm, 15 min, 4°C), filtered and RNase T1 was added at final concentration of 1 unit/µl for 10 min at 22°C. 20 µl beads (Dynabeads Protein G, Thermo Fisher Scientific) per 1 ml lysis buffer were washed twice with lysis buffer and coated for 1 h with 0,25 µg/µl FLAG antibody (Sigma-Aldrich, F1804). Afterwards beads were washed with lysis buffer twice and added to the sample. After 2 h incubation at 4°C on a rotating wheel the beads were washed with IP wash buffer (50 mM HEPES-KOH [pH 7.5], 300 mM KCl, 0,05 % IGEPAL CA-630, 0,5 mM DTT, complete EDTA-free protease inhibitor cocktail) 3 times and a second RNase T1 treatment was carried out. For replicate 1 a final concentration of 1 unit/µl and for replicate 2 a final concentration of 15 unit/µl was used for 10 min at 22°C. Subsequently, beads were washed with high-salt was buffer (50 mM Tris-HCl [pH 7.4], 1 M NaCl, 1 % IGEPAL CA-630, 0,1 % SDS, 0,5 % sodium deoxycholate, 1 mM EDTA, complete EDTA-free protease inhibitor cocktail [Roche]) 3 times and 2 times with polynucleotide kinase buffer (50 mM Tris-HCl [pH 7.5], 50 mM NaCl,10mM MgCl2, 5 mM DTT). Samples were radiolabeled using T4 polynucleotide kinase (NEB) and resolved on a Novex 4-12% Bis Tris gel (Thermo Fischer Scientific). The protein-RNA complexes were transferred to a nitrocellulose membrane (Whatman), exposed to a phosphorimager screen for 30 min and excised at approximately 160 kDa. After proteinase K (Roche, 40 min at 50°C) digest the RNA was extracted by phenol-chloroform treatment and precipitation. small RNA cloning and library generation (Hafner et al.,2012, PMID:22926237) using 3′ adaptor 4N-RA3, 5′ adaptor OR5-4N, RT primer RTP and PCR primers RP1 (forward primer) and RPI1-12 (reverse primer, containing barcodes, for sequences see Illumina Truseq small RNA oligonucleotides)