GSM5994425: AH_8h3h_n1; Homo sapiens; RNA-Seq
Total RNA was isolated using the RNeasy Plus Mini Kit according the instructions with the modification that dithiothreitol (DTT) was added in all washing steps (0.1 mM) and to the final eluate (1 mM). Afterwards, extracted total RNA was alkylated using 10 mM iodoacetamide (in 50 mM sodium phosphate buffer, pH 8, 50% DMSO). After 15 minutes incubation at 50°C, the reaction was quenched by addition of 20 mM DTT, followed by subsequent ethanol precipitation. cDNA libraries were generated with the QuantSeq 3' mRNA-Seq Library Prep Kit FWD with Illumina with the UMI Second Strand Synthesis Module for QuantSeq (Lexogen). Library sequencing (single-read; 75 or 150 cycles) was performed on a NextSeq 500 sequencer using a High Output Kit v2.