GSM8325682: WT_CD45+Immune_7M; Mus musculus; RNA-Seq
CD45+ enriched immune cell fraction: Briefly, minced kidney were digested in the digestion buffer for 20 minutes at 37°C, passed through a 70-micron cell strainer, and collected by centrifugation. Cells were resuspended in blocking solution (PBS without calcium and magnesium containing 0.04% BSA and Fc blocking solution) and incubated for 30 minutes on ice. Cells were then washed with PBS with 0.04% BSA and incubated for 30 minutes at room temperature with the PE rat anti-mouse CD45 (1:200, catalog no. 12–0451, 30-F11; eBioscience). After washing, the cell suspension was sieved through a 40-micron cell strainer and centrifuged at 4°C. Cells were also stained with viability dye (DAPI). Samples were sorted using a BD FACS Aria II. Sequencing libraries were constructed according to 10x standard protocol by following the steps cDNA fragmentation, end repair and A-tailing, size selection by SPRIselect beads (Beckman Coulter), adaptor ligation, sample index PCR amplification, and repeat SPRIselect beads size selection. The final constructed single-cell libraries were sequenced by Illumina Novaseq machine with total reads per cell targeted for a minimum of 30,000.