The Chromium Next GEM single cell 3′ HT reagent kits v3.1 (user guide CG000416, 10X Genomics, Inc.) were used for the single-nuclei RNA-seq assay. The single nuclei suspension from a pool of 10 donor tissue samples was loaded into two wells of a Chromium Next GEM chip M to target 60,000 cell recovery per well. The chip was run on a Chromium X (10x Genomics). Single-cell gel beads in emulsion containing barcoded oligonucleotides and reverse transcriptase reagents were generated. cDNA was synthesized and amplified following cell capture and cell lysis. The quality and quantity of cDNA and resulting libraries were examined by Bioanalyzer. The final libraries were sequenced on an Illumina NovaSeq 6000. 100-bp reads, including cell barcode and UMI sequences, and 100-bp RNA reads were generated.
