GSM5531883: Comma-1D - scRNAseq; Mus musculus; RNA-Seq
The Comma-1D cell line was thawed from frozen aliquots and cultured in CytoOne T25 flasks (USA Scientific) with culture media composing DMEM/F-12 media (Thermo Fisher), 2% FBS (Sigma-Aldrich), 1% Pen-Strep (Gibco), 10 µg/ml Insulin (Sigma-Aldrich), and 5ng/ml Endothelial Growth Factor (Thermo Fisher). Upon 80% confluency, the sample was passaged following the recommended sub culturing protocol for adherent cells. For single-cell RNA sequencing, cells were collected at passage 5 and suspended in PBS media at 1 x 106 cells/mL. Cells was processed according to Chromium 3' Gene Expression V3 Kit (10X Genomics) using manufacturer's guidelines followed by sequencing on a S1 NovaSeq chip (Illumina Inc.). Qubit 3 (Fisher Scientific) and 2100 Bioanalyer (Agilent) were used for quality check of cDNA. The output BAM file from sequencing was processed through 10X Genomics Cell Ranger software v3.1.0. This outputs a read counts matrix that we used for downstream analyses using Seurat, a customizable R-Studio Package for scSeq analysis.