GSM2483505: PBT003 gliblastoma stem cells, control shRNA, m6A-IP; Homo sapiens; RIP-Seq
Total RNA was extracted by Trizol (Ambion) according to the manufacturer's instructions. mRNA was then purified using Dynabeads mRNA purification kit (Ambion, Catalog # 61006). RNA fragmentation was performed by sonication at 10 ng μl-1 in 100 μl RNase-free water using Bioruptor Pico (Diagenode) with 30 s on / 30 s off cycle for 30 cycles. 5% of the fragments was saved as input. m6A-immunoprecipitation (m6A IP) and library preparation were performed according to a published protocol (Dominissini et al., 2013). In detail, 2.5 μg affinity purified anti-m6A rabbit polyclonal antibody (Synaptic Systems; Catalog # 202003) and 20 μl Protein A beads (ThermoFisher; Catalog# 10002D) were used for each affinity pull-down. m6A antibody-bound RNAs were eluted with 100 μl elution buffer and recovered by RNA Clean and Concentrator-5 (Zymo), and subjected to RNA library preparation with TruSeq Stranded mRNA Library Prep Kit.
GEO Accession
GSM2483505