GSM5832727: AB29: HEK-293 Borr 72h - rep2; Homo sapiens; RNA-Seq
RNA extraction was performed with the RNeasy Mini Kit (74104, Qiagen) including DNase I on-column digestion (1010395, Qiagen) and elution in 50 µl RNase-free water. The library for RNA-seq was prepared with 200 ng RNA input and the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England BioLabs (NEB), E7760L). First, mRNA was enriched using the NEBNext Poly (A) mRNA Magnetic Isolation Module (NEB, E7490L) and following the manufacturer's instructions. Briefly, after isolation, samples were fragmented for 15 min at 94°C for a target insert size of 200 bp. Next, the first and the second cDNA strand were synthetized, followed by an Ampure XP bead (Beckman Coulter, A63881) clean up. Then the samples were end-prepped and the adaptor was ligated. The adaptor was diluted following the manufacturer's instructions. The adaptor-ligated samples were purified with Ampure XP beads before amplification. A different Unique Dual Index Primer Pair (NEB, E6440S) was added to each sample and 11 cycles were used for the PCR enrichment. Purification using Ampure XP beads was used to obtain the final library. The quality and quantity of the library was assessed with the D1000 assay on the Tapestation (Agilent, 5067-5582 and 5067-5583) and the dsDNA HS assay from Qubit (Thermo Fisher, Life Technologies, Q32851).