title: GSM7831828: IRd21-Choline, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: The kidneys were enzymatically digested in PBS supplemented with 5% fetal bovine serum (FBS), 0.25% trypsin and 10μg/mL lDNase I. For around 40 minutes, the renal tissues were subjected to cell dissociation at 37 °C and 50 r.p.m shaking. To maximize the yield and viability of cells, they were collected at 20-minute intervals during the dissociation process, ensuring optimal recovery and preservation. After filtration through a 40 μm nylon cell strainer, the cell suspensions were treated with 1x red blood cell lysis solution  to remove red blood cells. Then, 1x DPBS supplemented with 2% FBS was used to wash the dissociated cells. The vitality of cells was determined by staining them with 0.4% Trypan blue  and counting them with the Automated Cell Counter. In a Gel Beads-in-emulsion (GEM) system, beads with cell barcodes and unique molecular identifiers (UMIs) were loaded almost to saturation, ensuring a one-to-one pairing of each cell with a bead. After being exposed to a cell lysis buffer, polyadenylated RNA molecules hybridized to the beads. For reverse transcription, all of the beads were placed in a single tube. At the 5' end of cDNA synthesis, which corresponds to the 3' end of the messenger RNA transcript, each cDNA molecule was labelled with UMIs and cell labels to specify its cellular origin. The 10x beads were then subjected to universal amplification, adaptor ligation, and second-strand cDNA synthesis. Through randomly interrupted whole-transcriptome amplification, sequencing libraries were created in order to enrich the 3' end of transcripts connected with the cell barcode and UMI.