GSM9180715: untreated POP92 cell line, replicate 2; Homo sapiens; RIP-Seq
Protein G Dynabeads were washed twice and resuspended in antibody conjugation buffer (1x PBS, 2mM EDTA, 0.1% BSA (w/v). 5 µg of anti-dsRNA mAb (J2) (SCICONS, cat# 10010500) were bound to 30 µl of washed beads overnight at 4° C on a rotating wheel. 107 POP92 cells per IP were fixed with 0.1% paraformaldehyde at room temperature (RT) for 10 min. Immediately, cells were quenched by adding glycine and washed twice with cold PBS. Crosslinked cells were lysed in lysis buffer (20mM Tris [pH 7.5], 150mM NaCl, 10mM EDTA, 10% Glycerol, 0.1% NP-40, 0.5% Triton X-100, supplemented with protease inhibitor tablet) for 15 minutes on ice. Following a spin at 12,000g at 4° C for 15 minutes, supernatant was transferred to a new eppendorf. The lysate was then immunoprecipitated using 30 µL antibody-conjugated Dynabeads per IP reaction overnight at 4° C in a rotator. Following magnetic separation, beads were washed three times with high salt wash buffer (20 mM Tris pH 7.5, 500 mM NaCl, 10 mM EDTA, 10% glycerol) and resuspended in 1X TBS. Per IP, 2 µL of Promega RNasinPlus RNase inhibitor (Fisher Scientific, PRN2611) and 0.5 µL of proteinase K (NEB, P8107S) was added. Decrosslinking was performed for all the IP samples at 65° C for 15 minutes. The Direct-zol RNA MiniPrep kit (Zymo Research, R2051) was used to extract RNA from IP supernatant. Samples were treated with Turbo DNase to remove any DNA contamination in the extracted RNA. Library prep was performed using Illumina Stranded total RNA ligation with Ribo Zero plus according to the manufacturers protocol.