GSM5348488: DUS3L with 5FUrd treatment 3; Homo sapiens; RIP-Seq
Cells were washed twice with cold PBS and then 500 µl lysis buffer (50 mM Tris–HCL pH 7.4, 100 mM NaCl, 1% NP-40, 0.1% SDS and 0.5% sodium deoxycholate) was added to each dish. Lysates were treated with Turbo DNase (AM2239) and a low (1:200) or high (1:50) concentration of RNaseI (AM2295) at 37 °C for 3 mins with rotation after which they were centrifuged at 13000  rpm for 15 min at 4°C. The supernatant was incubated with anti-FLAG beads (200 μl Protein G beads and 15 µg anti-Flag M2 antibody) at 4 °C overnight with rotation, and the immunoprecipitated material was then washed 3 times with high-salt buffer and then once with CutSmart buffer (NEB) washing. The beads were treated with Quick-CIP (NEB) at 37 °C for 30 mins with rotation, washed twice with high-salt buffer, and then incubated with preadenylated and biotin labeled L3 linker by using T4 RNA ligase I (NEB) at 16 °C overnight. After washing 3 times with high-salt buffer, the beads were boiled with 60 μl sample buffer and 3 μl was analyzed by Western blot for biotinylation with the chemiluminescent nucleic acid detection module (Thermo). The rest of the sample was gel purified based on the position of the biotin signal. Immunoprecipitated RNA was recovered using D-tube dialyzer midi (Merck-Millipore) and digested with proteinase K (Roche). Reverse transcription was performed using oligonucleotides containing randomized barcodes (UMIs) and two inversely oriented adaptor regions separated by a BamHI restriction site. cDNAs were size-purified (70-80nt, 80-100nt and 100-150nt) on TBE-Urea gels and then circularized by CircLigase II (Epicentre). Circularized cDNAs were digested with BamHI and linearized cDNAs were amplified using Solexa primers and submitted for Illumina sequencing.