GSM6805763: Notch3, scRNAseq; Mus musculus; RNA-Seq
Euthanized mice were perfused with chilled 1× PBS via the left ventricle. Kidneys were harvested, minced into approximately 1 mm3 cubes and digested using Multi Tissue dissociation kit (Miltenyi, 130-110-201). The tissue was homogenized using 21G and 26.5G syringes. Up to 0.25g of the tissue was digested with 50μl of Enzyme D, 25μl of Enzyme R and 6.75 μl of Enzyme A in a ml of RPMI and incubated of 30 minutes at 37°C. Reaction was deactivated by 10% FBS. The solution was then passed through a 40 μm cell strainer. After centrifugation at 400g for 5 minutes, cell pellet was incubated with 1ml of RBC lysis buffer on ice for 3 minutes. Cell number and viability were analyzed using Countess AutoCounter (Invitrogen, C10227). This method generated single cell suspension with greater than 80% viability. 10,000 cells were loaded into the Chromium Controller (10X Genomics, PN-120223) on a Chromium Single Cell B Chip (10X Genomics, PN-120262) and processed to generate single-cell gel beads in the emulsion (GEM) according to the manufacturer's protocol (10x Genomics, CG000183). Libraries were generated using Chromium Single Cell 3' Reagent Kits v3 (10x Genomics, PN-1000092) and Chromium i7 Multiplex Kit (10x Genomics, PN-120262) according to the manufacturer's manual. Quality control for constructed library was performed by Agilent Bioanalyzer High Sensitivity DNA kit (Agilent Technologies, 5067-4626) for qualitative analysis. Quantification analysis was performed by Illumina Library Quantification Kit (KAPA Biosystems, KK4824). The library was sequenced on Illumina HiSeq 4000 system with 2 × 150 paired-end kits using the following read length: 28 bp Read1 for cell barcode and UMI, 8 bp I7 index for sample index, and 91 bp Read2 for transcript.