title: GSM8791690: Non-treated bladder muscosa 1; Mus musculus; RNA-Seq
library_construction_protocol: Separated mucosa was minced into small pieces and then digested at 37oC in a buffer containing 1 mg/mL Collagenase IV and 100 ug/mL DNase I in PBS for 3o minutes. After the incubation period, the digestion solution was triturated using a 5 mL pipette for 1 minute before centrifuging for 10 minutes at 350xg at 4oC. The supernatant was then removed, and the pellet was resuspended in 700 uL of accutase (STEMCELL Technologies, 07922). The suspension was incubated in a nutator mixer for 10 minutes at 37oC, which was then centrifuged for 10 minutes at 350xg at 4oC. Next, the pellet was resuspended using 0.5 mL of RBC lysis buffer containing NH4Cl, NaHCO3 and EDTA in Millipore water and incubated for 1 minute before adding 4.5 mL of PBS to stop the RBC lysis. The suspension was then passed through 70 um cell strainer into a different 50 mL tube and centrifuged for 10 minutes at 350xg at 4oC. The supernatant was then removed and 300 uL of cell staining buffer (PBS with 2% FBS) was added to the cells. The cells were sorted using fluorescence-activated cell sorting and propidium iodide (PI) (1:300) staining was used to sort the live cell. Following the manufacturer's protocol, Chromium Single Cell 3' Reagent Kit v3.1 (10x Genomics) was used to perform single-cell RNA sequencing. In short, a single-cell suspension was prepared ensuring >90% cell viability using 1% BSA-PBS. The concentration of cells was adjusted to 700-1000 cells/uL and capture of ~10,000 cells per sample was targeted. To encapsulate single cells with barcoded Gel Beads, cells were loaded into Chromium Chip B and the Chromium Controller was used to generate Gel Beads-in-Emulsion (GEMs). Further, to generate barcoded cDNA, reverse transcription was implemented within GEMs and the cleanup was performed after using silane magnetic beads. PCR was performed to amplify the barcoded cDNA, and the product purification was done using SPRIselect magnetic beads. Then, fragmenting the cDNA, end repairing, A-tailing and ligating sequencing adapters were performed to construct the sequencing libraries. The libraries were purified using SPRIselect beads after another round of PCR amplification and quantification was performed using Tapestation 4150 (Agilent G2992AA). The 10x Genomics gene expression libraries were pooled together and sequenced on an Illumina NovaSeq 6000 system using an S1-100 flowcell. Each library contained ~10,000 cells and 53,000 reads per cell were targeted. Paired-End sequencing with a configuration of read 1 = 28bp, read 2 = 90bp, dual-index = 10bp was performed and 1% of phiX control library was added to the flowcell to serve as a sequencing control.