GSM9789674: NC_3; Homo sapiens; RNA-Seq
A total of 10 μg total RNA from each sample was used to prepare a directional RNA-seq library. First, the polyadenylated mRNAs were concentrated with oligo (dT)-conjugated magnetic beads (Invitrogen; Thermo Fisher Scientific, Inc.). The concentrated mRNAs were then iron-fragmented at 95˚C, end-repaired and ligated to a 5′ adaptor. RT was performed with RT primer harboring a 3′ adaptor sequence and randomized hexamer. The purified cDNAs were amplified and stored at -80˚C until they were used for sequencing. Following the manufacturer's instructions, the libraries were prepared for high-throughput sequencing. The Illumina Novaseq 6000 (Illumina, Inc.) was used to collect data from 150-bp pair-end sequencing (Illumina, Inc). Raw sequencing reads containing more than 2-N bases were first discarded. Subsequently, the raw reads were trimmed of adaptors and low-quality bases using a FASTX-Toolkit (v.0.0.13; http://hannonlab.cshl.edu/fastx toolkit/).