title: GSM8888376: Urethra, Adult mus female, rep 1; Mus musculus; RNA-Seq
library_construction_protocol: Whole urethras were dissected and washed with HBSS to remove fat and debris. Cells were chopped and digested in Accutase supplemented with DNaseI and 10 uM Rock inhibitor Y-27632 for 1.5 hours. Cells were triturated to generated single suspensions and passed through a 30 micron filter to obtain single cells. Following RBC lysis, cell counts were obtained using a hemocytometer. Single cells were loaded onto the Chromium controller for single cell partitioning and barcoding. For single cell isolation of the epithelial and stromal compartments, female urethras were cut into two pieces each digested in a 1% trypsin solution containing DNaseI. Epithelial tissue was mechanically separated from the stromal tissue. Tissues were separately digested to single cells using Accutase as described above for the whole urethras. A target of 10,000 cells per sample was loaded on to the 10x Chromium contoller for cell partitioning. Libraries were prepared according to the manufacturers instructions for the 10x Genomics Chromium Next GEM Single Cell 3' Kit v3.1. Briefly, cells were resuspended in the master mix and loaded along with the partitioning oil and gel beads onto the Chromium Next GeM Chip G to generate gel bead-in emulsions (GEM). Poly-A mRNAs captured by the beads were reverse transcribed to cDNA along with the unique molecular identified (UMI), Illumina R1 primer and the cell barcode. Following reverse transcriptions, GEMs were broken up and cDNAs were pooled. Silane magnetic beads were used to purify first strand cDNA from the RT mixture. Full length cDNA was amplified by PCR. Enzymatic fragmentation and size selection was performed to obtain desired read lengths. Illumina R2 primer, P5 and P7 bridge amplification primers and sample index is introduced using end-repair, A-tailing, ligation and PCR. Librariers were pooled for sequencing.