GSM8188180: 22_0500_TM; Homo sapiens; RNA-Seq
Primary hTM cells were isolated from human donor corneal rims deemed unsuitable for transplantation, and characterized. All donor tissues were procured from Saving Sight eye bank (Kansas city, MO). All donor cells used in this study were isolated (between 2012 and 2021), validated, stored in liquid nitrogen, or used in prior studies from our lab. Prior to single cell sequencing, frozen cells were thawed with 15ml pre-warmed media (37C, DMEM:F12=1:1, with 20%FBS, 1%Penn/Step/fungizone) and centrifuged at 300g, 4C for 5min. Cells were washed with media again and suspended in 0.04% BFA and ready for single cell RNA sequencing. Cell viability was checked with trypan blue. Resuspended single cells were loaded on a 10X Chromium controller for obtaining single cell Gel Beads-In-Emulsions. scRNA-seq libraries were generated using 10X Chromium Single Cell 3' reagent kits v3.1 (10X Genomics) following the manufacturer's recommendations (https://www.10xgenomics.com). Sequencing was performed on Illumina Novaseq 6000 (http://www.illumina.com) at the Single Cell Genomics Core at Baylor College of Medicine.