GSM8137144: BRX394-DRAW-1_S22; Homo sapiens; RNA-Seq
Prior to extraction, live cells were separated from dead cells using Ficoll-Paque Plus (Cytiva) enrichment by centrifuging at 400×g for 5 minutes at room temperature and removing the top layer and washing it with PBS. RNA was isolated from live cells using the Quick-RNA Microprep Kit (Zymo). Purified RNA was resuspended in 4μl of Mix-1, denatured at 72°C for 3 min and transferred to ice for 1 min before 7μl of Mix-2 was added. Reverse transcription was carried out at 50°C for 90 min, followed by 5 min incubation at 85°C.  cDNA was then purified with Agencourt AMPureXP SPRI beads (Beckman Coulter), to remove all primer dimer residues. cDNA concentration measurements were performed using the Qubit dsDNA high sensitivity assay kit on the Synergy H1 Hybrid Microplate Reader (BioTek). Libraries were generated using the Nextera XT Library Prep kit (Illumina) with custom indexing adapters in a 384-well PCR plate, followed by a cleanup step to remove residual primer dimers. Combined libraries from different cells were then sequenced on a NextSeq 500 sequencer (Illumina), using paired-end 38-base reads.