title: GSM8652197: VHKO Female 2; Mus musculus; RNA-Seq
library_construction_protocol: Kidneys were bisected, the renal capsule removed, and bisected kidneys were macerated for up to 7 min on a bed of ice. Macerated kidneys were then subjected to single-cell dissociation using the Multi-Tissue Dissociation Kit 2 (Miltenyi 130-110-203). Briefly, macerated tissues were suspended in 1.45 ml Buffer X, 30 µl of Enzyme D, 15 µl of Enzyme P, 15 μl of Buffer Y, and 6 µl of Enzyme A, all prepared according to the manufacturer's instructions. The suspension was then incubated under water at 37°C for 30 min in a shaking incubator set to 150 rpm. Dissociation was stopped by the addition of 150 µl Fetal Bovine Serum (FBS; Sigma F7524) and resuspension in 9 ml of RPMI-1460 medium (Merck R0883). The digest was filtered through a 40 µm cell strainer to remove undigested tissue and the filtrate was centrifuged (300g for 10 min at 4°C). Erythrocytes were eliminated by resuspending dissociated cells in 3 ml of 1x RBC Lysis Buffer (Miltenyi 130-094-183) prepared in deionized water, and incubating for 2 min at RT. Cells were centrifuged (300g for 5 min at 4°C) and resuspended in ice-cold D-PBS before being counted on a Thermo Scientific Countess II machine for total yield and viability. Sorted cells were prepared into single-cell droplets using the Chromium Next GEM Single-cell kit. 20,000 live cells were loaded per sample on separate Chromium Next GEM Chip G (10X Genomics PN-1000127) channels. cDNA clean-up, amplification and adaptor ligation were performed with the Chromium Next GEM Single Cell 3ʹ Kit v3.1 (10X Genomics PN-1000268). cDNA yield was quantified using the High Sensitivity D1000 ScreenTape Assay (Agilent 5067-5584 and 5067-5585) to optimize the number of reaction cycles for library preparation. Single cell sequencing libraries were prepared using the Library Construction Kit (10X PN-1000190) and Dual Index Kit TT Set A (10X Genomics PN-1000215) sequence indices. Single-cell libraries were sequenced on Illumina NextSeq 2000 sequencing system with P3 200 cycle (Illumina 20040560) reagents. Libraries from up to six samples were pooled in an equimolar ratio, diluted to 650 pM in RSB buffer and mixed with 1% PhiX Control v3 DNA (Illumina FC-110-3001) for sequencing. Libraries from up to six samples were pooled in an equimolar ratio, diluted to 650 pM in RSB buffer and mixed with 1% PhiX Control v3 DNA (Illumina FC-110-3001) for sequencing.