GSM7518280: Parietal cells, MGI0900 72hr; Mus musculus; RNA-Seq
Fresh gastric corpus was harvested and mechanically dissociated in DMEM media+ Collagenase 1A while shaking at  37° for 30 minutes. Tissue was further dissociated chemically in 5-15 minutes at 37° in 0.05% trypsin-EDTA. The single cell suspension was passed through a 30µm filter, quantity and viability assessed with hematocytometer, and submitted to the Washington University Genome Technology Access Core (GTAC) in 0.04% BSA 1x PBS. Library was performed according to the manufacturer's instructions (single cell 3' v3.1 protocol, 10x Genomics GEX). Briefly, cDNA was prepared after the GEM generation and barcoding, followed by the GEM-RT reaction and bead cleanup steps.  Purified cDNA was amplified for 11-13 cycles before being cleaned up using SPRIselect beads.  Samples were then run on a Bioanalyzer to determine the cDNA concentration.  GEX libraries were prepared as recommended by the 10x Genomics Chromium Single Cell 3' Reagent Kits User Guide (v3.1 Chemistry Dual Index) with appropriate modifications to the PCR cycles based on the calculated cDNA concentration.  For sample preparation on the 10x Genomics platform, the Chromium Next GEM Single Cell 3' Kit v3.1, 16 rxns (PN-1000268), Chromium Next GEM Chip G Single Cell  Kit, 48 rxns (PN-1000120), and Dual Index Kit TT Set A, 96 rxns (PN-1000215) were used.  The concentration of each library was accurately determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer's protocol (KAPA Biosystems/Roche) to produce cluster counts appropriate for the Illumina NovaSeq6000 instrument.  Normalized libraries were sequenced on a NovaSeq6000 S4 Flow Cell using the XP workflow and a 50x10x16x150 sequencing recipe according to manufacturer protocol.  A median sequencing depth of 50,000 reads/cell was targeted for each Gene Expression Library.