title: GSM7745116: RNA-Seq - A549ACE2-EGFP-2; Homo sapiens; RNA-Seq
library_construction_protocol: Cells were lysed using Trizol reagent (Thermo Fisher) and RNA was extracted by phase separation. Total RNA was quantified by nanodrop (DeNovix DS-11) and TapeStation 4200 (RNA Screen Tape; Agilent Technologies, Inc) or 2100 Bioanalyzer (Total RNA Nano; Agilent Technologies, Inc). 500 ng of total RNA was used for library preparation via the KAPA mRNA HyperPrep kit (Roche) according to the manufacturer's recommendations. Briefly, mature mRNA was isolated from total RNA using mRNA Capture Beads. Following elution in Fragment, Prime and Elute buffer (FPE), RNA was fragmented using heat (6 min at 94oC) in the presence of Mg2+. First and second strand synthesis and A-tailing were performed according to manufacturer's recommendations. 7 μM KAPA Unique Dual-Indexed (UDI, Roche) adapters were ligated to the second strand synthesis product in the presence of a ligation master mix in a reaction that was performed at 20 oC for 15 min. Following cleanup, all libraries underwent 11 cycles of amplification. Successful library production, quality control, and quantification was assessed using Tapestation (High sensitivity D1000 Screen Tape, Agilent). Libraries were pooled together and subjected to NovaSeq 6000.