GSM5737765: fibroblasts, control, rep10; Homo sapiens; RNA-Seq
Total RNA was extracted from the treated primary fibroblasts using Agencourt RNAdvance Tissue kit (Beckman Coulter Life sciences, cat# A32646) and quantified using a Quant-iT RiboGreen RNA Assay Kit (Invitrogen, cat# R11490). 250 ng RNA was used to generate libraries using the QuantSeq 3' mRNA-Seq library prep kit (FWD) HT for Illumina (Lexogen). The kit uses total RNA as input, hence no prior poly(A) enrichment or rRNA depletion is needed. The libraries were amplified for 17 cycles quantified with Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, cat# P7589). Size pattern was controlled with the High Sensitivity NGS Fragment Analysis kit on a Fragment Analyzer (Agilent DNF-474). Libraries were pooled at an equimolar ratio (i.e., an equal quantity of each sample library) and clustered at a concentration of 9 pM on single read (SR) sequencing flow cell (Illumina). Sequencing was performed for 65 cycles on a HiSeq 2500 (Illumina) using the Rapid SBS Kit v2 (Illumina, cat# FC-402-4021). Sequencing reads were trimmed with BBDuk (BBTools version 35.85, Bushnell B., sourceforge.net/projects/bbmap/).