GSM4160974: HV4_CTR_3: Huvec Isolate #4 - DMSO (R3); Homo sapiens; RNA-Seq
RNA was isolated using RNeasy Mini Kit (Qiagen). Approximately 500ng Total RNA was used for RNA-seq library preparation by following the Illumina TruSeq stranded mRNA sample preparation guide. The first step in the workflow involves purifying the poly-A containing mRNA molecules using poly-T oligo-attached magnetic beads. Following purification, the mRNA is fragmented using divalent cautions under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. This is followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. Strand specificity is achieved by replacing dTTP with dUTP in the Second Strand Marking Mix (SMM). These cDNA fragments then go through an end repair process, the addition of a single 'A' base, and then ligation of the adapters. The products are then purified and enriched with PCR to create the final RNA-seq library. After RNA-seq libraries were subjected to quantification process, pooled for cBot amplification and subsequent 50bp single read sequencing run with Illumina HiSeq 3000 platform.
GEO Accession
GSM4160974