GSM8948035: DB21_P-SF3B1_T211_OTS964_REP1; Homo sapiens; RIP-Seq
Described in detail in library construction protocol iCLIP was performed as previously described (Huppertz et al., 2014) with only minor modifications described below. Each cell pellet (originally from one 150 cm2 plate) was lysed in 1 ml of lysis buffer and the lysate was homogenized by passing three-times through an insulin syringe. Lysate was treated with 4 U/ml Turbo DNase (Thermo Fisher, AM2238), 12 U/ml RNase I (Thermo Fisher, AM2295) shaking at 1100 rpm and 37°C for 3 min. Clarified extracts (21000g for 30 min) were incubated for 2 h with appropriate antibody pre-bound to 50 µl of protein G Dynabeads. After series of stringent washes, adenylated L3 RNA adapter was ligated to the 3'end of crosslinked RNAs. Proteins and crosslinked RNAs were resolved by SDS-PAGE (NuPAGE 4-12% Bis-Tris Protein Gel, Thermo Fisher Scientific, NP0322) and transferred to nitrocellulose membrane. The region of the membrane containing the radioactively labeled crosslinked protein–RNA complexes was excised, RNA was isolated and reverse transcribed to cDNA. cDNA was fractionated using urea denaturing gel electrophoresis and three fractions running between 70-85 nt (L-low), 85-120 nt (M-medium) and 120-200 nt (H-high) were isolated. Each fraction was independently circularized by single-stranded DNA ligase, annealed to an oligonucleotide complementary to the restriction site and cut between the two adapter regions by BamHI. After final PCR amplification using P3 and P5 Solexa primers all three fractions were pooled together in ratio 1:5:5 (L:M:H). Multiplexed libraries were sequenced as 75 bp single-end reads on Illumina sequencer (HiSeq 2500 or NextSeq 500)