GSM8137374: A3_P3_82_NT; Homo sapiens; RNA-Seq
Prior to extraction, live cells (determined by live/dead dye) were flow sorted into 96 wells with lysis buffer using the Smart-Seq2 protocol (Illumina). 96-well plates containing cell lysates were thawed on ice, spun down at 1500 rpm for 30s, and mixed with Agencourt RNAClean XP SPRI beads (Beckman Coulter) for RNA purification. 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 minincubation at 85°C. 14μl of Mix-3 was added in each well and the whole-transcriptome amplification step was performed at 98°C for 3 min, followed by 21 cycles at (98°C for 15 s, 67°C for 20 s and 72°C for 6 min), and final extension at 72°C for 5min. cDNAwas 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 adapters64in 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.