GSM8605728: Male term diabetic (T-D), scRNAseq, rep 1; Mus musculus; RNA-Seq
The kidneys were directly perfused via the left ventricle with 0.9% saline. The kidneys were rapidly dissected, moved to ice-cold PBS, then minced on ice using a scalpel. Each minced kidney was transferred into a 15 mL centrifuge tube with 2.0 mL of the protease solution for each half of the kidney (protease solution: ice-cold: 25 mM CaCl2, 10 mg/mL B. Licheniformis protease, 125 U/mL DNase I, 1xDPBS). Samples were digested on ice and triturated using p1000 tip for 15 seconds every 2 minutes for a total of 20 minutes. The digestion was stopped by adding 3 mL of ice-cold PBS + 10% FBS and centrifuged at 1200g for 5 min at 4°C. The pellet was resuspended in 2 mL of PBS + 10% FBS, and cells were filtered through a 70-μm cell strainer and rinsed with 2 mL of PBS + 0.01% BSA. Next, cells were centrifuged and resuspended in 5 mL of PBS +0.01% BSA. Cells were filtered the second time through a 40-μm cell strainer and rinsed with 2 mL of PBS + 0.01% BSA, centrifuged, and resuspended in 1 mL of 1x RBC Lysis Buffer. Red blood cell lysis was performed for 5 minutes with occasional shaking, at room temperature. The reaction was stopped by adding 20 mL of 1xPBS, cells were centrifuged at 1200g for 5 min at 4°C and resuspended in PBS + 0.04% BSA. Cell suspension was stained with Propidium Iodide (Thermo Fisher #P1304MP), and live cells were sorted using the BD FACSMelody cell sorter. Sorted cells were centrifuged at 1200g for 5 min at 4°C and resuspended in 50μl of PBS + 0.04% BSA. Samples were processed and prepared for scRNA sequencing at University of Virginia, School of Medicine University of Virginia - UVA Genome Analysis and Technology Core (RRID:SCR_018883, 5P30CA044579). Single-cell suspensions were introduced into a multi-channel microfluidics chip within the Chromium Controller (10x Genomics Inc., Pleasanton, CA, USA) to generate Gel Beads in Emulsion (GEMs), aiming for a cell recovery target of 5,000 to 10,000 cells. The single-cell suspension was processed using the 10x Genomics Chromium Single Cell System (10x Genomics, Inc.). Gene expression libraries were prepared with the Chromium™ Next GEM Single Cell 3' Kit v3.1, 16 rxns (PN-1000268), the Chromium™ Next GEM Chip G Single Cell Kit, 48 rxnt (PN-1000120), and the Chromium™ Dual Index Kit TT Set A 96 rxns (for 3' and 5' Gene Expression, TCR/BCR and Multiome) (PN- 1000215).  A small portion of the prepared library was sequenced on the MiSeq to perform initial quality control, including checking cell count, assessing the quality of the library, and performing rapid cluster analysis, later the sequencing was performed on the NextSeq 500 sequencer. Raw sequence data were processed using CellRanger (10x Genomics, Inc) and Bcl2fastq (https://support.illumina.com), generating sample-specific FASTQ files. The fastq files were aligned to the mm10 (version 2020-A) reference transcriptome using the 'cell ranger count' function from Cell Ranger pipeline (v 7.0.1), resulting in a total of 17,326 cells estimated across all samples (3,727 cells in T-ND, 3,810 in PT-ND, 5,065 in T-D, and 4,742 in PT-D).