GSM7697101: Glomeruli young rep2; 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. For bulk RNA sequencing, glomeruli were placed directly into RNeasy Lysis Buffer (Qiagen). RNA was extracted and isolated from samples using RNeasy Mini Kit (74104, Qiagen) according to manufacturer instructions. Regular input RNA-Seq was performed as follows. Total RNA was quantified with Qubit RNA HS Assay Kit (Thermo Fisher Scientific) and quality was assessed using RNA ScreenTape on 4200 TapeStation (Agilent Technologies). For sequencinglibrary generation, the Truseq Stranded mRNA kit (Illumina) was used with an input of 100 nanograms of total RNA. Libraries were quantified with Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and the average library size was determined using D1000 ScreenTape on 4200 TapeStation (Agilent Technologies).Libraries were pooled and sequenced on NovaSeq 6000 (Illumina) to generate 30 millions single-end 50-base pairreads for each sample.