GSM5501366: dbm2_S1_L001; Mus musculus; RNA-Seq
The mice were anesthetized and perfused with chilled 1×PBS via the left heart. Kidneys were removed, minced into 1 mm3 pieces with a razor blade, and incubated in 2 ml of digestion buffer containing 1mg/ml collagenase type II (Sigma), 1 mg/ml Pronase E (Boster), and 50μg/ml DNase I ( Roche) at 37℃ for 10 minutes. The partially digested tissue was then passed through a 100-μm cell strainer (Falcon) into HBSS (Hank's Balanced Salt Solution) twice on ice and pelleted by centrifugation (200g for 5mins). Then, the pellet was resuspended with HBSS and sheared with a 27G needle to remove large clumps. They were further filtered through a 40-μm cell strainer (Falcon) to get single cell solution. After centrifugation, the cell pellets were incubated with 3ml of RBC lysis buffer (TIANGEN, RT122-02) to remove red blood cells at room temperature for 3 minutes. Finally, cells were washed and resuspended in HBSS for three times to remove debris or cell aggregates. Cell viability and number was determined using Countess® II Automated Cell Counter (Invitrogen, C10227) after stained with Trypan blue. Using this method, we were able to generate single cell suspensions with > 80% viability. Single cells were resuspended with HBSS to a suitable concentration (1600,000-7000,000 cells/ml). The viable cells were then loaded into Chromium microfluidic chips with 3' (Gel Bead Kit version 2) chemistry and barcoded with a 10x Chromium Controller (10xGenomics). RNA from the barcoded cells was subsequently reverse-transcribed to generate cDNA and sequencing libraries constructed with reagents from a Chromium Single Cell 3'v2 reagent kit (10x Genomics) according to the manufacturer's instructions. Sequencing was performed with Illumina (NovaSeq 6000) using 150bp paired-end sequencing.