GSM5389327: 10X scRNA-seq HNES1 naive hPSC; Homo sapiens; RNA-Seq
Chromium Single Cell 3′ Library & Gel Bead Kit v3 Single-cell RNA was isolated from individual cells, harvested and dissociated with 0.25% trypsin (10min at 37°C) resuspended in 0.1% BSA in DMEM/F12, and processed with a Chromium Single Cell 3′ Library & Gel Bead Kit v3 and Chromium Chip B Kit Single-cell RNA-seq libraries were prepared using the following: Chromium Single Cell 3′ Library & Gel Bead Kit v3 (10x Genomics, PN-1000075), Chromium Chip B Kit (10x Genomics, PN-1000073) and Chromium Single Cell 3' Reagent Kits v3 User Guide (Manual Part CG000183 Rev C, 10X Genomics). Cell suspensions were loaded on the Chromium instrument with the expectation of collecting gel-beads emulsions containing single cells. RNA from the barcoded cells for each sample was subsequently reverse-transcribed in a C1000 Touch Thermal cycler (Bio-Rad) and all subsequent steps to generate single-cell libraries were performed according to the manufacturer's protocol with no modifications. cDNA quality and quantity were measured with Agilent TapeStation 4200 (High Sensitivity 5000 ScreenTape) after which 25% of material was used for gene expression library preparation. Library quality was confirmed with Agilent TapeStation 4200 (High Sensitivity D1000 ScreenTape to evaluate library sizes) and Qubit 4.0 Flourometer (ThermoFisher Qubit™ dsDNA HS Assay Kit to evaluate dsDNA quantity). Each sample was normalized and pooled in equal molar concentration. To confirm concentration, pool was qPCRed using KAPA Library Quantification Kit on QuantStudio 6 Flex before sequencing. Pool was sequenced on S2 flowcell on Illumina NovaSeq6000 sequencer with following parameters: 28 bp, read 1; 8 bp, i7 index; and 91 bp, read 2.