title: GSM6870672: D0, replicate 1, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Freshly collected kidney cells from glyoxylate treated mice were transported in sterile culture dish with 10 ml 1X Dulbecco's Phosphate-Buffered Saline (DPBS, Thermo Fisher, 14190144) on ice to remove the tissue storage solution, then minced on ice. Tissues were digested with 0.25% Trypsin (Thermo Fisher, 25200-072) and 10 µg/ml IDNase I (Sigma, 11284932001) dissolved in PBS with 5% Fetal Bovine Serum (FBS, Thermo Fisher, SV30087.02). Tissues were dissociated at 37℃ with a shaking speed of 50 rpm for about 40 minutes. Cells were repeatedly collected at interval of 20 minutes to increase cell yield and viability. Cell suspensions were filtered using 40 µm nylon cell strainer and red blood cells were removed by 1X Red Blood Cell Lysis Solution (Thermo Fisher, 00-4333-57). Dissociated cells were washed with 1 X DPBS containing 2% FBS. Cells were stained with 0.4% Trypan blue (Thermo Fisher, 14190144) to check the viability on Countess® II Automated Cell Counter (Thermo Fisher). Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.