GSM3324337: WT.d4.IP.2 (RIP_miRNA); Homo sapiens; ncRNA-Seq
Cell pellets from approximately 50 million cells were homogenized, lysed in ice-cold lysis buffer (10 mM HEPES [pH 7.3, ThermoFisher Scientific], 100 mM KCl [1049360250, Merck], 0.5 % NP40 [NP40S, Sigma-Aldrich], 5 mM MgCl2, 0.5 mM dithiothreitol [DTT, DTT-RO, Sigma-Aldrich], protease inhibitors [118735, Sigma-Aldrich], recombinant RNase inhibitors [10777019, ThermoFisher Schientific], 1 mM PMSF [36978, ThermoFisher Scientific]) using TissueLyser LT (50 Hz, 2 min, Qiagen). For lysate clearing, homogenates were centrifuged for 15 min at 16,200 × g, 4°C. A 50 µl sample was taken as a pre-IP sample. The RIP samples were incubated with Dynabeads Protein G beads (10009D, ThermoFisher Scientific) coated with anti-hAGO2 antibody (a gift from Jay Nelson; Grey et al., 2010) for 24h at 4°C with end-over-end rotation. After incubation, beads were collected on a Dynamagnet (1 min, on ice, ThermoFisher Scientific) and gently resuspended in low-salt NT2 buffer (50 mM Tris-HCl [pH 7.5, T1503, Sigma-Aldrich], 1 mM MgCl2, 150 mM NaCl [S3014, Sigmal-Aldrich], 0.5 % NP40, 0.5 mM dithiothreitol, 1 mM PMSF, protease inhibitors, recombinant RNase inhibitors). The beads were transferred to a new collection tube and washed with low salt NT2 buffer, followed by two washes with high salt NT2 buffer (50 mM Tris-HCl [pH 7.5], 1 mM MgCl2, 600 mM NaCl, 0.5 % NP40, 0.5 mM DTT, protease inhibitors, 1 mM PMSF, recombinant RNase inhibitors). The solution containing the RNA was then transferred to Qiazol (Qiagen). Subsequently, RNA was isolated from RIP and pre-RIP samples according to the miRNeasy micro kit protocol (Qiagen). The size distribution of the samples and the quality was checked with a Bioanalyzer RNA 6000 Pico Chip (Agilent Genomics). cDNA libraries were prepared using NEXTFlex Small RNA Library Prep V3 (NOVA-5132-06, BIOO Scientific) with 50 ng input and 22 PCR cycles.
GEO Accession
GSM3324337