GSM7697108: Aged glomeruli rep1 [scRNA-seq]; Mus musculus; RNA-Seq
Mice were perfused transcardially with 10 ml of RPMI with 200 ul of M-450 Dynabeads (Thermo Fisher Scientific, 14013). Kidneys were immediately extracted and minced using a razor blade, then transferred to 5 ml digestion buffer with LiberaseTM (1.5 U/ml; 5401127001, Millipore Sigma) and DNaseI (100 U/ml; D4527, Millipore Sigma) in RPMI-1640, and incubated for enzymatic digestion (37⁰C, 800 rpm, 20 min). Following digestion, samples were sieved via a 100 μm and a 70 μm MACS® SmartStrainers (130-098-463, 130-098-462, Miltenyi Biotec), and 30 ml ice-cold 10% FBS in RPMI-1640 were added to stop digestion. Samples were washed twice with 30 ml ice-cold RPMI-1640 and glomeruli were isolated using a magnetic separator (DynaMag-2, Thermo Fisher Scientific, 12321D). Glomeruli were dissociated in 1 ml digestion buffer with Trypsin (0.5% wt/vol; Thermo Fisher Scientific, 15090046), Dispase II (2 U/ml; Roche, 04942078001), Collagenase D (2 U/ml; Roche, 11088866001), and DNAse I (10 U/ml; D4527, Millipore Sigma), in RPMI-1640, and incubated for enzymatic digestion (37⁰C, 800 rpm, 40-60 min). Samples were kept on ice until further processing. Cell counts and viability were determined before further analyses and viability was determined at >95% viable cells. Samples were kept on ice and immediately processed. Sample processing for scRNA-seq was done using Chromium Next GEM Single Cell 3' Kit v3.1 (1000269, 10X Genomics) according to manufacturer's instructions. Cell concentration was used to calculate the volume of single-cell suspension needed in the reverse transcription master mix, aiming to achieve approximately 8000 cells per sample. cDNA and libraries were prepared according to manufacturer's instructions (10X Genomics). Sequencing reads were processed through 10X Genomics Cell Ranger (v.3.1.0) and aligned to GRCm38.