GSM7730917: SV Rep 2; Homo sapiens; RNA-Seq
Fresh biopsy tissues were minced into 1-2mm pieces, digested overnight in collagenase solution, and further dissociated using 0.25% trypsin. After dissociation to single cells, cells were cultured in epithelial selection media (KSFM) until they reached 70-80% confluency (4-8 days).). Cells from each biopsy were passaged one time for expansion before freezing. Prior to the scRNA-seq experiment, the PrECs were thawed, passaged, and maintained in KSFM for 3-4 days until they reached 70% confluency. Cell partitioning, library preparation, and sequencing was performed in a single batch using the 10X Genomics platform. 10X gem generation and barcoding was performed according to the Chromium Next GEM Single Cell 3' Reagent Kits v3.1 User Guide Rev D for a targeted cell recovery of 5000 cells per sample (4 groups with two replicates each for 8 samples total). Post GEM-RT cleanup was performed on all samples according to the 10X protocol, followed by cDNA amplification (12 cycles), and quantification using Agilent TapeStation. 3' gene expression dual index library construction was performed using dual index plate TT set A, according to the 10X protocol.