GSM8018996: bladder_Tom_plus; Mus musculus; RNA-Seq
The bladder from a 25-month-old male p16-CreERT2-tdTomato mouse was harvested two weeks after consecutive five days i.p injection of tamoxifen. The harvested bladder tissue was minced using a blade and then subjected to agitation two times using 18 G and 21 G needles during one-hour incubation at 37℃ in 2ml PBS containing 5 mg Collagenase Type 4 (GIBCO) and 5 KU/ml of DNase I (Sigma-Aldrich). The cell suspension was filtered through a 70 µm strainer (Falcon), followed by centrifugation (800 x g, 4℃, 3 minutes). The pellet was then resuspended in a wash buffer (4% FBS in PBS). After pelleting the cells, they were resuspended in wash buffer containing 10% FcR blocking reagent (MACS) for 15 minutes. Subsequently, the cells were stained with an anti-CD45 antibody (Biolegend) for 30 minutes at 4°C in the absence of light. After pelleted, cells were resuspended in wash buffer containing with 0.1 µg/ml DAPI (Cell Signaling Technology) to exclude dead cells during sorting. The CD45-/DAPI-/Tomato+ and CD45-/DAPI-/Tomato- cells were sorted by using BD FACSAria SORP (BD Bioscience). For the scRNA-seq library construction, we followed the manufacturer's instructions of 10x Genomics Chromium Single Cell 3′ Reagent Kit v3.1.