title: GSM7999426: normal mice, replicate1, ScRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Mice were sacrificed by cervical vertebra dislocation and bladder was removed. The fresh bladders were washed with cold phosphate buffered saline (PBS), dissected, minced, and incubated in 20 mg/ml dispase II (sigma D4693), 1 mg/ml DNaseI (solarbio D8071) and 0.5 mg/ml Liberase TL (roche 5401020001) solution at 37°C for 2 hours. The digested tissue was homogenized by vigorous shaking and filtered through 100 μm cell strainer. Mononuclear cells were then harvested from the interphase of an 80% and 40% Percoll gradient after a spin at 2500 rpm for 20 min at room temperature. CD16/32 antibody (eBioscience) was used to block the non-specific binding to Fc receptors before CD45.2 (1:200, Biolegend 109808) antibody staining. The live and dead cells were discriminated by Live and Dead violet viability kit (Invitrogen). Fluorescent cell sorting was used to attain living CD45+ single-cell suspensions. Single-cell suspensions were loaded onto microfluidic devices, and single-cell RNA-sequencing (scRNA-seq) libraries were constructed according to the Singleron GEXSCOPE™ protocol in the GEXSCOPE™ Single-Cell RNA Library Kit (Singleron Biotechnologies). Pools were sequenced on Illumina novaseq6000 with 150 bp paired end reads. Raw reads were processed with fastQC and fastp to remove low-quality reads. Unique molecular identifiers (UMI) and cell barcodes were extracted after filtering out reads without poly-A tails. Poly-A tails and adapter sequences were then trimmed by cutadapt, and the reads that met quality control standards were matched to the reference genome mm10 using STAR.