GSM5277042: Cryo-hESC-CEC3; Homo sapiens; RNA-Seq
Total RNA was isolated from each sample using TRIzol reagent. The extracted RNA was examined using a NanoDrop Lite spectrophotometer and RNA 6000 Pico kit on an Agilent 2100 Bioanalyzer for RNA-Seq library prepration. Total RNA was subjected to RNA-Seq library preparation using NEBNext Ultra II RNA Library Prep Kit for Illumina. Approximately, 1.0 μg of high-quality total RNA (≥8.0 RIN Score) was used for polyadenylated RNA selection using oligo dT beads, followed by the fragmentation of selected polyadenylated RNA. The fragmented RNA was used as a template for cDNA synthesis by reverse transcriptase with random primers. The cDNA was further converted into double-stranded DNA that was end-repaired to incorporate the specific index adapters for multiplexing, followed by a purification step and amplification for 15 cycles. The amplified libraries were examined on an Agilent 2100 Bioanalyzer and quantitative PCR (qPCR) according to the manufacturer's instructions. RNA-Seq libraries with unique index sequences were pooled in an equimolar ratio, and a final size selection of 400–500 bp was performed. After the quality control confirmation, RNA-Seq bar-coded pooled libraries were sequenced using HiSeq 2500 (rapid run mode) genome analyzer.