GSM8242287: SYO.1, RNA-seq, Treated, Rep 2; Homo sapiens; RNA-Seq
Total RNA was extracted from the SYO-1 untreated and treated cells (with 100 nM TAK-981 for 36h) using the Isolate IIRNA Mini kit #BIO-52072, Bioline) Stranded mRNA-seq library preparation and next generation sequencing: Approximately 500ng Total RNA was used for stranded mRNA-seq library preparation by following the NEB Directional mRNA-seq sample preparation guide (New England Biolabs, Ipswich, MA). The first step in the workflow involved purifying the poly-A containing mRNA molecules using poly-T oligo-attached magnetic beads. Following purification, the mRNA was fragmented into small pieces using divalent cautions under elevated temperature. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. This was followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. Strand specificity was achieved by replacing dTTP with dUTP in the Second Strand Marking Mix (SMM). These cDNA fragments then went through an end repair process, the addition of a single 'A' base, and then ligation of the adapters. The products were then purified and enriched with PCR to create the final RNA-seq library