GSM7817714: STZ, replicate 3, scRNAseq; Mus musculus; RNA-Seq
Heart (ventricle), liver and kidneys were dissected and finely minced on ice using scissors. Cells were enzymatically dissociated in collagenase II (500U/ml; Thermo Fisher 17101015) in 10 ml HBSS, with agitation at 37°C for 1 hour. Dissociated heart, liver and kidney cells were passed through a 70 µm cell strainer and rinsed with 3 ml ice-cold HBSS. Spleens were passed through a 70 µm cell strainer into 1X Red Cell Lysis Buffer (1.5 mM ammonium chloride, 100 µM sodium bicarbonate, 11 µM EDTA disodium) and incubated for 10 minutes at room temperature. All cells were centrifuged for 5 min at 400 g at 4°C, then resuspended in 10% v/v DMSO in foetal bovine serum (FBS), aliquoted and frozen at -80ºC before sequencing. Cells were revived by thawing at 37 °C for 1 min in a water bath and slowly transferred into 10 ml tubes filled with 9 ml pre-warmed RPMI containing 10% FBS and centrifuged at 500 g for 5 min. Next, cells were resuspended in 500 µl RPMI containing 10% FBS and labelled with commercially available DNA-labelled antibodies (TotalSeq-A, Biolegend) at 4 °C for 30 min. Following incubation, cell suspensions were washed three times with PBS containing 2 % BSA. After the final wash, cell suspensions were resuspended in PBS containing 2 % BSA and a viability dye (Zombie Red, Biolegend) for discrimination between live and dead cells. Next, 10,000 viable cells were sort-purified from the four organs of each individual mouse using a SH800 cell sorter (Sony). The four cell fractions were pooled for processing as a single sample according to the manufacturer's protocol, while the antibody-linked barcodes enabled sample-specific demultiplexing of the sequencing data. scRNA-seq libraries were generated using the Chromium Controller and the Next GEM Single Cell 3' Reagent Kit (v3, 10x Genomics) according to the manufacturer's instructions. Libraries were sequenced by the Biomedical Sequencing Facility at the CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, using the Illumina NovaSeq 6000 platform.