GSM7967289: Sham, wildtype, kidney, scRNAseq; Mus musculus; RNA-Seq
All mice were perfused with 20 ml of  ice-cold PBS to remove remaining blood in the vasculature. Freshly collected organs (kidney or lung) were diced by scapels and razor blade and incubated with 40 U/ml of DNase (Sigma) and 50 microg/ml of Liberase (Roche) in RPMI media (gibco) for 60 minutes at 37°C in a shaking incubator. Digested organs were strained through 70 micron filters and then red blood cells were lysed with ACK lysis buffer. The cell mixtures were again strained through 40 micron filters. Aquired single cell suspensions were washed and re-suspended in PBS, stained with propidium iodide, washed and resuspended in FACS buffer (PBS, 0.5% BSA, 2mM EDTA, 0.01% NaN3), and live cells were sorted using FACS Aria III (BD Biosciences). Libraries were prepared using the Chromium Single Cell 5′ Library Kit v2 and Chromium instrument (10× Genomics, Pleasanton, CA, USA) according to manufacturer's instruction. Briefly, for the gel bead-in-emulsion (GEM) generation, single-cell samples were resuspended in the master mix and applied together with partitioning oil and barcoded gel beads onto the Chromium Chip. Incubation of the individual GEMs including cell lysate and reverse transcription (RT) reagents generated full-length cDNA with barcodes from polyadenylated mRNA in every single GEM. The RT products contained Illumina R1 primer sequence, Unique Molecular Identifier (UMI), template switch oligo (TSO), and the 10x Barcode. Then GEMS were broken, pooled, and purified with Silane magnetic beads. The full-length cDNA was amplified by PCR, followed by enzymatic fragmentation and size selection for library construction. During the library construction, P5, P7, i5 and i7 sample indexes, and Illumina R2 primer sequence were added.