Sequencing library construction using the 10x Chromium platform
We Approximately 15000-20000 cells were partitioned into nanoliter droplets to achieve single cell resolution for a maximum of 5000 individual cells per sample. The resulting cDNA was tagged with a common 16-nt cell barcode and 10- to 12-nt unique molecular identifier during the reverse transcription (RT) reaction. Full-length cDNA from poly A–tailed mRNA transcripts was enzymatically fragmented and selected to optimize the cDNA amplicon size (~400 bp) for library construction (10x Genomics). The single cell library concentration was quantified by quantitative PCR (qPCR) analysis (Kapa Biosystems) to produce appropriate cluster counts for the NovaSeq 6000 platform (Illumina). We generated 28–98 bp (3′ v3 libraries) or 2–150 bp (5′ libraries and TotalSeq libraries) sequence data targeting ~50 K read pairs/cell for the gene expression library, which provided digital gene expression profiles for each individual cell.