GSM5456121: ERKi 250nM 8h DOX Rep 1, S53; Homo sapiens; RNA-Seq
Cells were lysed with 600µl Trizol per well and total RNA was prepared using Direct-zol-96 RNA Kits according to the manufacturer's protocol. Sample concentrations were determined by Nanodrop and RNA quality was assessed on a subset of samples by Bioanalyzer (Agilent); all samples scored RINs of > 9.0. RNA sequencing library preparation was performed with the High Throughput TruSeq Stranded mRNA Library Prep Kit (Illumina) following the manufacturer's protocol at half reaction volume. Input for each sample consisted of 300-500ng of RNA and 5ul of 1:500 diluted ERCC spike-in mix 1 (Ambion). Libraries were amplified for 12 cycles during the final amplification step. Libraries were quantified using the Qubit dsDNA HS assay (Thermo Fisher Scientific). Library size and quality were spot checked for a subset of samples by Bioanalyzer (Agilent). The average size of cDNA fragments in the libraries was 370 base pairs. Libraries were pooled at equimolar concentrations then the pool was quantitated using the KAPA library quantification kit (KAPA Biosystems). Libraries were sequenced single end 114 base pairs using NovaSeq_SP full flow cell (Illumina) at the Bauer Core Facility (Harvard University).