title: GSM9300865: Renal pelvis-Wild type mouse; Mus musculus; RNA-Seq
library_construction_protocol: Adult C57BL/6 male mice were deeply anesthetized and transcardially perfused with chilled PBS. Thirty renal pelvises were dissected and temporarily stored in tissue preservation solution (Miltenyi Biotec, Cologne, Germany; 130-100-008). After single washes with PBS, samples were digested enzymatically at 37°C for 30 min using a digestion cocktail containing: 2 mg/mL collagenase I, 2 mg/mL collagenase II, 1 mg/mL collagenase IV, 2 mg/mL dispase II, and 50 U/mL DNase I in RPMI 1640 medium (Thermo Fisher Scientific; 11875093). The enzymatic reaction was quenched by adding an equal volume of RPMI-1640 medium. The cell suspension was centrifuged at 500 ×g for 6 min, resuspended in 0.25% Trypsin-EDTA (25200056, Thermo Fisher Scientific), and incubated for 5 min at 37°C. Subsequently, the digested suspension was filtered through a 70-μm cell strainer and centrifuged again at 500 ×g for 6 min. Cells were washed twice with PBS containing 0.03% BSA, and their viability was assessed as 94.2% using an automated cell counter (Countstar BioTech; ALIT Life Sciences, Shanghai, PR China). Single-cell suspensions were loaded onto a Chromium Controller instrument (10x Genomics, Pleasanton, CA) to generate gel bead-in-emulsions (GEMs) with the Chromium Single Cell 3' v3.1 Reagent Kit (10x Genomics, 1000268). Reverse transcription within GEMs was performed in a thermal cycler (SimpliAmp, Thermo Fisher Scientific) under the following program: 53°C for 45 min, 85°C for 5 min, and hold at 4°C. Barcoded cDNA was amplified, fragmented, a-tailed, and ligated with Illumina adaptors using a library construction kit (10x Genomics, PN-1000190). Libraries were sequenced on a NovaSeq 6000 system (Illumina, San Diego, CA) with a sequencing depth of ~48,902 reads per cell.