GSM3168222: OE Ago2 eCLIP-seq rep1; Homo sapiens; RIP-Seq
Total RNA was extracted by TRIZOL reagent. For Aog2 CLIP-seq, HeLa cells grown on 15 cm plates were first UV irradiated at 400 mj and collected by scraping the cells. After IP, the 3' RNA adapter was ligated to RNA fragments on beads, which was followed by reverse transcription and ligation of a 3' single-stranded DNA adapter to the cDNA. Libraries were sequenced by Hi-Seq 2500. For Ribo-seq, transfected cells were collected and resuspended with ice-cold lysis buffer (10 mM Tris-Cl pH7.4, 5mM MgCl2, 100 mM KCl, 1% Triton X-100) and placed on ice for 20 min. The cell lysate were centrifuged at 12000g for 20 min, and then the supernatant was loaded on a 10%-50% linear sucrose gradient and centrifuged at 36400 rpm for 3 hrs in SW41Ti rotor (Beckman). After centrifugation, ribosome and polyribosome fractions were collected and monitored by UV254. The collected fractions were pelleted by ultracentrifuge at 50,000 rpm in SW60 rotor for 6 hrs and ribosomal bound RNAs were digested with RNase I (Ambion). The RNA fragments were size selected (~30 nt) and rRNA depleted by antisense oligo and RNase H strategy. Ribosome protected fragments(RPFs) were ligated with the 5' and 3' adaptor and sequenced on Hi-Seq 2500. For RNA-seq, A fraction of total RNA from the same cells was each isolated using TRIZOL Reagent (Ambion) and fragmented using reverse transcriptase buffer (Invitrogen) at 94oC for 10 min. RNA fragments were size selected (180-220 nt) and rRNA depleted by antisense oligo and RNase H strategy, and then ligated with 5′ and 3′ adaptor. The libraries were sequenced on Hi-Seq X10.
GEO Accession
GSM3168222