GSM8943557: iMGL derived from major allele hiPSC treated with vehicle (replicate3); Homo sapiens; RNA-Seq
Cells were lysed using QIAzol Lysis Reagent (QIAGEN) for the extraction of RNA. The lysates were separated into an aqueous phase containing RNAs using chloroform (Daejung) and centrifuged at 12,000 rpm for 15 mins at 4°C in RNase-free condition. The RNA pellets were washed with RNase-free 75% ethanol, air-dried, and suspended in RNase-free water. Total RNA concentration was measured using Quant-IT RiboGreen (Invitrogen, #R11490), and RNA integrity was assessed using Agilent TapeStation RNA ScreenTape (#5067-5576). Only samples with RIN > 7.0 were used. Libraries were constructed using 1 µg of total RNA per sample with the Illumina TruSeq Stranded mRNA Library Prep Kit (#20020595). Poly-A containing mRNA was purified using poly-T magnetic beads, fragmented, and reverse-transcribed using SuperScript II reverse transcriptase (Invitrogen, #18064014). Second-strand cDNA synthesis was carried out using DNA Polymerase I, RNase H, and dUTP. The cDNA fragments were end-repaired, A-tailed, adapter-ligated, and PCR-amplified. Libraries were quantified using the KAPA Library Quantification Kit.