title: GSM5258591: WT Urothelium rep4; Mus musculus; RNA-Seq
library_construction_protocol: 5 month BBNK5VP16;Pparg and control bladders wereflash frozen sagitally in OCT and stored at-80°C. The specimen blocks were placed in the LeicaCM3050 cryostat chamber for 5-10min to temperature equilibrate. The tissue was sectioned at 5μm on Arcturus PEN Membrane glass slides (Applied Biosystems cat#LCM0522) and the slideswere immediately stored at-80°C. 1 hour prior to LCM, slides were removed from the-80°C andstained with Arcturus HistoGene Staining Solution (ThermoFisher Scientificcat#KIT0415)according to the manufacturer's protocol. Tissue was then laser captured on a ZeissAxioObserver.Z1 inverted microscope into AdhesiveCap 200 opaque caps (Zeiss cat#000830-19).240μL of RLT Lysis Buffer (Qiagen cat#1015750) was then added to the capturedtissue Cold active protease (CAP), was prepared and stored on ice (5mMCaCl2, Sigma cat#21115, 10mg/mL Bacillus Licheniformis protease, Sigma cat#P5459, 12.5U/mLDNAse, Sigma cat#4716728001). Mice were perfused with 20 mL of CAP using a small veininfusion set (Kawasumi cat#D3K2-23G) and two 10 mL syringes per mouse. Bladders weredissected and immediately put in a 60mm x 15mm petri dish (Fisherbrand cat#FB0875713A)containing CAP on ice for 10 min. The bladders were then transferred to HBSS media(ThermoFisher cat#14170-112) containing 1% Bovine Serum Albumin (Sigma cat#A2058) and0.1% glucose, where they were inverted and the urothelium was manually separated from thestroma. The cell suspension was then collected into a 1.5mL Eppendorf tube on ice and gentlytriturated until the cells were in a single cell suspension The libraries were prepared using the SMART-Seq® v4Ultra® Low Input RNA Kit for Sequencing (TaKaRa) followed by Nextera XT (Illumina), bothaccording to manufacturer's instructions