Briefly, animals were perfused with 8ml bead solution and 2ml bead solution with enzymatic digestion buffer (Collagenase type II 300U/ml, Proetainse E 1mg/ml and Dnase I 50U/ml), which was pre-warmed at 37°C. At the end of perfusion, kidneys were removed, decapsulated, minced into 1 mm3 pieces, and digested in 3ml digestion buffer at 37 °C for 15min on a rotator (100 r.p.m). Digested tissue was then passed through a 100-μm cell strainer and collected by centrifugation. The pellet was resuspended in 2 ml of Hanks’ buffered salt solution and glomeruli were washed three times and collected using a magnet. The separated glomeruli were resuspended in 2 ml digestion buffer and incubated at 37 °C for another 40 min at 1400 r.p.m/min on a thermomixer. During the digestion period, the solution was vortexed every 10 min and sheared with a 27G needle every 15 min. Then, the solution was put on magnetic particle concentrator and supernatant was pooled. The suspension was then sieved through 40 um cell strainer and centrifuged at 1500 r.p.m for 5 min at 4°C.  After two-step approach for primary cell purification, single cells were resuspended in 0.5ml of HBSS supplemented with 2% fetal bovine serum, 25mM HEPES and 4'6-diamidino-2-phenylindole (1 mg/ml). Total RNA was isolated from either isolated glomeruli or sorted podocytes by using RNeasy mini kit (Qiagen 74104) according to the manufacturer’s protocol. RNA concentrations were quantified using a Nano-drop Spectrophotometer at a wavelength of 260nm. RNA samples were then analyzed by Bioanalyzer at a concentration of 100-200 ng/ul to verify the concentration and the purity of samples. Only the samples with RNA integrity (RIN) values of >7.0 were used for mRNA sequencing at the Genomic Core Facility at Icahn School of Medicine at Mount Sinai School.
GEO Accession
GSM2090945