GSM9326116: Kidney, Tcf21, KO, Rep1; Mus musculus; RNA-Seq
1.5 kidneys from 4 male mice (2 Tcf21-KO mice, 1 Cre+ ctrl, and 1 Cre- ctrl) were harvested to obtain kidney single cell suspensions. Kidneys were minced with a razor blade and then digested in 0.3 mg/mL liberase TM, 10 µg/mL hyaluronidase, and 20 µg/mL DNAseI at 37°C x 40 min in an Eppendorf Thermomixer. The resulting cell suspension was treated with RBC lysis buffer and then incubated with 0.05% trypsin EDTA at 37°C x 30 min with shaking. 10% FBS in 1X PBS (without calcium/magnesium) was added to inactivate the trypsin. Cells were pelleted and resuspended in 0.01% BSA in 1X PBS (without calcium/magnesium). The suspension was then sequentially filtered through 100 µm and 40 µm strainers. Finally, cell number and viability were deternined by AOPI fluorescent staining using the Nexcelom Cellometer Auto2000. To recover 20,000 cells per sample, 29,000 cells from each of the 4 samples were loaded into the Chromium X Controller (10X Genomics, PN-1000328) on a Chromium GEM-X Single cell 3' Chip Kit v4 (10x Genomics, PN-1000215). Samples were processed to generate single cell gel beads in emulsion (GEM) according to the manufacturer's protocol. The cDNA and libraries were generated using the GEM-X Single cell 3' Kit v4 (10x Genomics, PN-1000691) and Dual Index Kit TT Set A (10X Genomics, PN-1000215) according to the manufacturer's manual. Quality control for constructed libraries was performed by the Agilent Bioanalyzer High Sensitivity DNA kit (Agilent Technologies, 5067-4626) and Qubit DNA HS assay kit for qualitative and quantitative analyses, respectively.