title: GSM8072572: RIP_IP; Homo sapiens; RIP-Seq
library_construction_protocol: Total RNA from tumor tissues or cell lines was extracted with TRIzol (Invitrogen). Total RNA was reverse transcribed using 4× Reverse Transcription Master Mix (EZBioscience, USA) according to the manufacturer's instructions. After total RNA was extracted, rRNAs were removed to retain mRNAs and ncRNAs. The enriched mRNAs and ncRNAs were fragmented into short fragments by using fragmentation buffer and reverse transcripted into cDNA with random primers. Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP(dUTP instead of dTTP) and buffer. Next, the cDNA fragments were purified with QiaQuick PCR extraction kit, end repaired, poly(A) added, and ligated to Illumina sequencing adapters. Then UNG(Uracil-N-Glycosylase) was used to digest the second-strand cDNA. The digested products were size selected by agarose gel electrophoresis, PCR amplified, and sequenced using Illumina HiSeqTM4000 by Gene Denovo Biotechnology Co. (Guangzhou, China). The libraries were sequenced by Illumina (Illumina, USA) with paired-end.