GSM2774582: HCT116_J2_Aza (v2); Homo sapiens; RNA-Seq
The 5-Aza-CdR treated cells were lysed with lysis buffer (30 mM HEPES-KOH at pH 7.4, 20mM MgOAc, 5mM DTT, 150mM KOAc, 0.1% NP-40, protease inhibitors). RNase T1(single-strand-specific RNA endonuclease) was added into the lysis buffer. The cell lysates were used for dsRNA pull-down with 3μg of J2 antibody (Scicons) on Dynabeads Protein G (Invitrogen) for 2 hours at 4°C. The beads were washed with wash buffer (30 mM HEPES-KOH at pH 7.4, 20mM MgOAc, 5mM DTT, 150mM KOAc, 0.1% NP-40, protease inhibitors). Digestions of single-stranded RNA were carried out by RNase T1 (Roche). Digestions of DNA were carried out by TURBO DNase (Thermo Fisher). dsRNA was purified and fragmented into 200 bp by shearing (Covaris). cDNA library preparation. RNA-seq libraries were prepared by using the NEBNext Multiplex Small RNA Library Prep Set for Illumina.