GSM3324380: WT.d0.IP.3 (RIP_mRNA); Homo sapiens; RNA-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 of three independent WT whole-cell RNA, ∆miR-124 whole-cell RNA, WT-AGO2 RIP, and ΔmiR-124-AGO2 RIP samples for 0 and 4 dpi were prepared using NEBNext Ultra I Directional RNA Library Prep Kit for Illumina (E7420, NEB) with 900 ng input. PolyA selection, fragmentation, first strand and second strand cDNA synthesis, and purification were performed in combination with Agencourt AMPure Kit (A63880, Beckman Coulter) and repair/dA-tailing of the cDNA. Adapters were ligated to dA-tailed cDNA and size-selected using AMPure XP Beads (Beckman Coulter). RIP samples were not polyA-selected. Index primers (Illumina) were used to index library constructs, and a PCR was performed using NEBNext Q5 2X PCR Master Mix (M0543S, NEB). Libraries were purified using Agencourt AMPure Kit. Libraries were pooled and sequenced on a HiSeq 2500 (Illumina), resulting in ca. 33-56 million single-end 75 bp reads.
GEO Accession
GSM3324380