GSM2299568: RWPE1 ERG-WT_RNA-seq_replicate 3; Homo sapiens; RNA-Seq
Total RNA from three biological replicates of RWPE1 cells expressing ERG-S96A was isolated using RNeasy kit (Qiagen) according to manufacturer’s protocol. Total RNA was DNase treated with RNase-Free DNase set (Qiagen) according to manufacturer’s protocol. polyA containing RNA was purified using oligo(dT) beads (Invitrogen). cDNAs of polyA selected RNA was generated with SuperScript III reverse transcriptase enzyme using random hexamers (Invitrogen). Second strand synthesis from cDNAs was carried out with E. coli DNA ligase and E. coli DNA polymerase I (New England BioLabs). The double-stranded cDNAs were sheared to ~150 nucleotide fragments using a Diagenode BioRuptor and the size was confirmed by DNA gel electrophoresis.Sheared DNA was end-repaired and ligated to indexing primers provided in Truseq sample preparation kit (Illumina). DNA libraries were pooled and sequenced in Illumina NextSeq 500 according to manufacturers protocol.