title: single cell RNA sequencing of bladder in germ-free and specific pathogen free mice
design: single cell RNA sequencing of bladder in germ-free and specific pathogen free mice
library_construction_protocol: Animals were euthanized and bladders were excised and kept on ice in Hypothermosol solution. Bladders were removed, and minced with sterile scissors and 2ml of Digestion solution was added (Ca/Mg-free PBS, 0.5mM EDTA pH8, 10mg/ml of Bacillus Licheniformis psychrophilic protease). Samples were incubated on ice for 20 min. Every minute they were flicked to mix, and every 2 min they were triturated using 1000 μl pipette with a cut tip. Then samples were split to three 1.7ml tubes and further incubated to 8 minutes, while homogenized with plastic pestle 10 strokes, every 2 min. Then samples were mixed thoroughly and centrifuged for 30s at 500g at 4 °C and sample was divided to 70% of top supernatant fraction, which was filtered trough 100-μm mesh, 5ml of Ca/Mg-free PBS was added and set aside on ice. The pellet was digested again. 1ml of Digestion solution was added and every minute they were flicked to mix, and every 2 min they were triturated using 1000 μl pipette with a cut tip. Then, the sample was filtered through 100-μm mesh, 5ml of Ca/Mg-free PBS was added and it was combined with first fraction. Cells were collected by centrifugation for 7 min at 500g at 4 °C. Then the pellet was resuspended in 300 μl of ACK buffer, incubated at room temperature for 1 min. Subsequently, 13 ml of cold Ca/Mg-free PBS was added, and samples were centrifuged at 500g 7 min at 4 °C. The cells were then incubated for one hour on ice in 100 μl Miltenyi Dead Cell Removal kit beads, after which 3 ml of kit Binding Buffer was added to each sample. The samples were then loaded onto LS-columns and the flow-through was collected. To increase cell recovery, columns were washed 3 times with 12ml of Binding Buffer. The flow through was then centrifuged 7 min 500g and was resuspended in Ca/Mg-free PBS with 0.04% BSA and samples proceeded directly to scRNAseq. according to 10x genomics Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 User Guide according to 10x genomics Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 User Guide