GSM5213527: E.1uM; Homo sapiens; RNA-Seq
To harvest the cells the cultures were washed twice with DMEM/F12 medium to remove dead cells and cell debris, dissociated with accutase for 12 minutes and collected in ice-cold DMEM/F12 containing 10 µM Rock-inhibitor. The cells were pelleted at 300 x g for 5 minutes at 4oC, resuspended in ice-cold DMEM/F12 containing glutamax, 0.04% BSA and 10 µM Rock-inhibitor and gently triturated using a 5 ml glass pipette. The cell suspensions were strained through a 40 µm filter, the viability of the harvested cells was approximately 90% for all samples as determined with trypan blue exclusion assay using an automated cell counter (Cellometer AutoT4 from Nexcelome Bioscience, Lawrence, MA). The cell concentrations were adjusted to 550 cells/µl and approximately 10,000 cells per channel (to give estimated recovery of 5,000-8,000 cells per channel) were loaded onto a Chromium Single Cell 3′ Chip (10x Genomics, PN-2000168) and processed through the Chromium controller to generate single-cell gel beads in emulsion (GEMs). scRNA-seq  libraries were prepared with the Chromium Single Cell 3′ Library & Gel Bead Kit v.2 (10x Genomics, PN-1000075).  Each sample was ligated and barcoded prior to performing library QC.  The Agilent Bioanalyzer and QuantStudio 12K Flex were used to assess the library quality prior to sequencing. Libraries from different samples were pooled based on molar concentrations and sequenced on a NovaSeq 6000 instrument (Illumina) with 151 bases for read 1, 151 bases for read 2 and 8 bases for index 1. After the first round of sequencing, libraries were re-pooled on the basis of the actual number of cells in each and re-sequenced to give an equal number of reads per cell in each sample and to reach a sequencing saturation of at least 50%. Chromium Single Cell Gene Expression