GSM6195593: D24_NACL_scRNAseq; Homo sapiens; RNA-Seq
Cells were washed with 1X PBS before dissociation from the culture dish by TryplETM Express Enzyme (ThermoFisher, 12605-036) incubation for 8 minutes at 37°C. Single cell suspension was filtered through a Falcon 40 µm Cell Strainer and centrifuged at 200G for 5 min. After resuspension in 1X PBS with 0.04% BSA, the single cell suspension was counted with a Luna-FL automated Fluorescence Cell Counter (Logos Biosystems). TSCs were mixed with primed and naive hPSCs using the following ratio (H9 primed: Sigma primed: H9 naive: Sigma naive: Sigma TSCs = 1:1:1:1:2) Cells were loaded onto the 10X Chromium Single Cell Platform (10X Genomics) targeting 2000 cells (Next GEM Single Cell 3' library and Gel Bead Kit v3.1) according to the manufacturer's protocol (10x User Guide; CG000204, Revision D). Generation of gel beads in emulsion (GEMs), barcoding, GEM-RT cleanup, complementary DNA amplification and library construction were all performed according to the manufacturer's protocol. Individual sample quality was assessed using a Tapestation (Agilent). Qubit 2.0 (ThermoFisher Scientific) and KAPA Library Quantification Kit for Illumina Platform (KAPA Biosystems) were used for library quantification before pooling. The final library pool was sequenced on the NovaSeq6000 (Illumina) instrument using NovaSeq SP kit (Illumina) for 2 lanes of 100-base-pair paired-end reads.