snRNA-seq of human lung
Nuclei were isolated from human snap frozen lung tissue using the Chromium10x genomics nuclei isolation kit, per manual. An aliquot of nuclei was stained with 0.4% trypan blue stain and DAPI and subsequently counted with the Thermo Fisher countess II FL. Samples with > 90% viability were used for downstream analysis. Approximately 20,000 nuclei per sample were loaded onto chip M and processed with the Chromium X controller. Barcoding and DNA (cDNA) library preparation was performed according to the manufacturers protocol (Chromium Next GEM Single Cell 3' HT Kit v3.1). Barcoding and DNA (cDNA) library preparation was performed according to the manufacturers protocol (Chromium Next GEM Single Cell 3' HT Kit v3.1). Quality of the cDNA was assessed at 2 time points with the Agilent Bioanalyzer High sensitivity DNA chip. There were 9 samples out of the 164 total experiments that we ran that were not of sufficient quality to proceed with sequencing. The cDNA libraries were sequenced on the Novaseq, using a Hiseq paired end 100bp sequencing configuration, with a read depth of approximately 60,000 reads and 3400 UMI per nuclei.