GSM5172769: Coculture; Homo sapiens; ssRNA-seq
Cells were lifted for flow cytometry with Accutase, and resuspended in PBS (Ca2+/Mg2+ free) supplemented with 0.5% BSA and 5μM DAPI. Dead cells were gated out based on DAPI staining. For single cell sequencing, two samples were prepared of live cells from CMs and EpiCs monocultured or cocultured. Two cellular suspension (monoculture and coculture) were loaded into two separate wells (estimated loading of 6,000 cells/well) in a 10X Chromium instrument using the Single Cell 3' reagent kit according to the manufacturer's instructions. MiSeq analysis confirmed greater than 90% of droplets contained cells compared to empty droplets, so we continued with paired-end sequencing performed on Illumina NovaSeq6000. Each fraction generated approximately 390 million raw reads which corresponded to approximately 71,000 reads per cell.