GSM8322275: VL-17A cells, control, rep 4; Homo sapiens; RNA-Seq
Cells were collected at the end of the experiment in RLT buffer (Qiagen) for RNA extraction. RNA was extracted using RNeasy mini kit (Qiagen) following manufacturer's instructions. DNase treatment was performed to remove genomic DNA. Libraries were prepared from 500ng of each total-RNA sample using the NEBNEXT Ultra II Directional RNA Library Prep kit (NEB #7760) and the Poly-A mRNA magnetic isolation module (NEB #E7490) according to the provided protocol. Poly-A containing mRNA molecules were purified using poly-T oligo attached magnetic beads. Following purification, the mRNA was fragmented using divalent cations under elevated temperature and primed with random hexamers. Primed RNA fragments were reverse transcribed into first strand cDNA using reverse transcriptase and random primers. RNA templates were removed and a replacement strand synthesised incorporating dUTP in place of dTTP to generate ds cDNA. AMPure XP beads (Beckman Coulter, #A63881) were used to separate the ds cDNA from the second strand reaction mix, providing blunt-ended cDNA. Multiple indexing adapters were then ligated to the ends of the ds cDNA to prepare them for hybridisation onto a flow cell, before 10 cycles of PCR were used to selectively enrich DNA fragments that had adapter molecules on both ends and amplify the amount of DNA in the library suitable for sequencing. Libraries were purified using AMPure XP beads, quantified by fluorometry using the Qubit dsDNA HS assay and assessed for quality and fragment size using the Agilent Bioanalyser with DNA HS Kit (#5067-4626).