snRNA-seq of homo sapiens: DIV35 vitro cells
Nuclei were thawed in ice and transferred in a glass douncer with 1mL of nuclei lysis buffer till complete homogenization.The nuclei suspension was centrifuged at 500 g for 5 min at 4 C, and resuspended in cold resuspension buffer. The resuspended nuclei were then filtered through a 30 m nylon mesh into pre-coated 1.5ml eppendorfs. DRAQ7 was added immediately before fluorescence-activated nuclear sorting. Intact nuclei were identified based on DRAQ7 and side scatter (SSC) signals and sorted with the BD FACSAria III Cell Sorter using a 100 m nozzle into collection tubes coated with D-PBS supplemented with 2% BSA. For snRNA-seq, 10,000 to 12,000 nuclei per sample (in up to 45 l total volume) were collected for the library preparation. Single nuclei were captured using the 10X Genomics Chromium system. After encapsulation with barcoded beads, lysis and reverse transcription were done using polyT primers carrying cell-specific barcodes, UMIs, and sequencing adaptors. The resulting cDNA was amplified. Library quality was checked with a Bioanalyzer, and sequencing was performed on an Illumina NovaSeq 6000 in 100 bp paired-end mode.