GSM6752572
Glomeruli were enriched by depleting the PT segment from the kidneys using Percoll density gradient centrifugation [12]. Both kidneys from each mouse were harvested, and the renal papilla of each kidney was removed. The renal tissue was minced into small pieces and transferred to a C tube (130-093-237, Miltenyi Biotec) filled with 5 ml of collagenase solution (1 mg/ml collagenase from Clostridium histolyticum [C9263, Sigma-Aldrich] and 0.25% bovine serum albumin [BSA] in DMEM/F-12 medium [11320074, Gibco]). The renal tissue was incubated at 37 °C for 5 min and homogenized using a gentleMACS™ Dissociator (Miltenyi Biotec) with the program “m. spleen 01_01”; this process was repeated once. Subsequently, the renal tissue was passed through a 250 µm sieve and separated by Percoll density gradient centrifugation using a solution that contained 45% Percoll® (GE17-0891-02, GE Healthcare) and 55% 2× PBS-glucose at 17,500 rpm for 15 min at 4 °C. Red blood cells were removed with a Percoll density gradient. PTs that accumulated in the interphase of the gradient were discarded. The rest of the kidney tissue that accumulated in the upper phase of the gradient was taken, washed twice in ice-cold HBSS and centrifuged at 400 ×g for 4 min at 4 °C. A single-cell suspension was prepared using CAP as described in previous studies [38]. The pellet was suspended in 2 ml of CAP solution (10 mg/ml Bacillus licheniformis protease [P5380, Sigma-Aldrich], 5 mM CaCl 2 , 20 U/ml DNase [4716728001, Roche] in PBS) for 20 min on ice with repeated trituration steps for 20 s every 5 min. The digestion was neutralized by 15 ml PBS supplemented with 10% FBS and passed through a 40 μm Corning® cell strainer. Cells were centrifuged at 400 ×g for 4 min at 4 °C and washed twice in 20 ml PBS supplemented with 0.5% BSA. Dead cells were removed with a dead cell removal kit (130-090-101, Miltenyi Biotec) according to the manufacturer's protocol. The living cells were passed through a 30 µm cell strainer (04-004-2326, Sysmex), and the cell concentration was determined using a TC20™ automated cell counter (Bio-Rad). The cells were loaded onto a 10x Genomics Chromium single-cell instrument. All steps were performed according to the standard protocol of the Chromium single-cell 3' v2/v3 kits to generate high-quality cDNA libraries.