GSM4759282: Pool16_19; Homo sapiens; RNA-Seq
For each pool, we generated a single cell suspension in 0.04% PBS-BSA and loaded approximately 10,500 single cells to the 10X Chromium Controller with a targeted recovery of 6,000 cells. ScRNA-seq libraries were generated using the 10X Genomics Chromium Single Cell 3' Kit v2 and the 10x Chromium Controller (10x Genomics) according to the 10X Single Cell 3' v2 protocol.  Single cells, reagents and single gel beads containing barcoded oligonucleotides were encapsulated into nanoliter-sized droplets and subjected to reverse transcription. Droplets were broken and the barcoded cDNAs were purified with DynaBeads and amplified by 12 cycles of PCR (98°C for 45 s; [98°C for 20 s, 67°C for 30 s, 72°C for 1 min] x 12; 72°C for 1 min). The amplified cDNA was fragmented, end-repaired, ligated with index adaptors, and size-selected with clean-ups between each step using the SPRIselect Reagent Kit (Beckman Coulter). Quality control of the resulting barcoded libraries was performed with the Agilent TapeStation and by PCR with primers specific to the P5 and P7 sequence (NEBNext Library Quant Kit for Illumina, New England Biolabs). Final 3' scRNA-Seq libraries were diluted to 4 nM, denatured, and further diluted to a concentration of 2.8 pM for sequencing with the following sequencing parameters: Read 1: 26 cycles, i7 index: 8 cycles, i5 index: 0 cycles, Read 2: 58 cycles on the NextSeq 500 (Illumina).
GEO Accession
GSM4759282