GSM8015446: Sorted renal cells, KO, Early, Male, tdTomato Negative, Rep 3; Mus musculus; RNA-Seq
Kidneys were dissociated using the Multi-Tissue Dissociation Kit 2 (Miltenyi 130-110-203). Briefly, the renal capsule was removed, and the tissue macerated for 5 min on a bed of ice. The macerated kidney was then transferred to 1.45 ml Buffer X, 30µl 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 tube was then agitated under water at 37˚C in a shaking incubator at 150 rpm for 30 min. 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). Undigested tissue was removed using a 40 µm cell strainer and the filtrate 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 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. Dissociated cells were resuspended in 10% FBS 2 mM EDTA in D-PBS (Thermo 14190144) to a concentration of 5 million live cells per ml. 10 μl of 10 μg/ml DAPI (Sigma D9542) was added to stain for viability. Cells were sorted using a BD Aria Fusion Cell Sorter. tdTomato was excited with a 561 nm laser and fluorescence detected with a 582/15 band pass filter. DAPI was excited with a 405 nm laser and fluorescence detected with a 450/40 band pass filter. Live, single, tdTomato-positive and -negative cells were collected in FBS-coated polypropylene tubes and pelleted by centrifugation at 300g for 10 min. Cells were counted again for yield and viability and processed for single-cell sequencing. 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). Twelve reaction cycles were used for cDNA amplification. 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 Dual Index Kit TT Set A (10X Genomics PN-1000215) sequence indices. Prepared libraries were quantified with KAPA Library Quantification Kit (Roche KK4824) following the manufacturer's protocol.