title: GSM5547766: KI SLAM-seq AS rep 1; Homo sapiens; OTHER
library_construction_protocol: RNA isolation was performed in the dark, following instructions in the SLAMseq Kinetics Kit – Anabolic Kinetics Module (Lexogen, 061.24) with the following modifications: frozen samples were heated for 5 min at 65 °C and, after addition of 200 µl chloroform:isoamylalcohol mix (24:1), further incubated for 15 min at 65 °C under vigorous shaking and repeated vortexing. The aqueous phase was separated by centrifugation and RNA was precipitated overnight at -20°C. Washing of the RNA pellet was performed twice with 1ml and 0.18 ml 75% ETOH (plus reducing agent), respectively. For resuspension in H2O, RNA was incubated for 15 min at 55 °C and 10 min on ice and repeatedly pipetted up and down. RNA concentration and quality was determined by capillary electrophoresis using the RNA 6000 nano kit (agilent, 5067-1511) and bioanalyzer (agilent). 4.5 µg RNA was alkylated by addition of Iodoacetamide following the manufacturer's instructions, but enhancing solubilization of the purified RNA by 15 min incubation at 55 °C. After quality control via capillary electrophoresis (RNA 6000 nano kit (agilent, 5067-1511), cDNA libraries were prepared from 500 ng RNA using the QuantSeq 3' mRNA-Seq Library Prep Kit for Illumina (FWD) (Lexogen, catalogue 015.24) and 16 PCR cycles for library amplification. DNA concentration and molarity were determined using the Qubit dsDNA HS Assay Kit (Thermo Fisher, Q33230) and Bioanalyzer High Sensitivity DNA Kit (5067-4626), respectively. 4 nM Libraries were pooled and sequenced using the NextSeq 500/550 High Output Kit v2.5 (Illumina) performing single end sequencing with 150 bp reads.