GSM7016387: Replicate 5, scRNAseq; Homo sapiens; RNA-Seq
The segmental/subsegmental fibrothrombotic material isolated from CTEPH patients were cut into ~ 3 mm pieces and digested using collagenase A for 1 hour. Cells were strained using 70 µM cell strainer. To remove red blood cells, cells were washed with ice cold DPBS and incubated with ACK (red blood cells lysis buffer) on ice for 5 mins. Cells were washed with DPBS and resuspended in cation-free HBSS that contains 10% FBS, 3 mM EDTA and 10 mM HEPES. Viable cells were counted manually using a hemocytometer. Cells were washed in DPBS and stained with Aqua (cell viability dye) (1:500). The stained cells were washed with ice cold DPBS and fixed with 80% of methanol for 15 mins. The viable cells were sorted using flow cytometry. After rehydration of methanol-fixed cells, scRNAseq libraries were prepared using the 10X Genomics Single Cell Protocol in collaboration with the Duke Human Vaccine Institute Genetics Analysis Core. Live cells were barcoded with cell-specific barcodes and unique molecular identifier (UMI) based on the Chromium Single Cell 3' library with Gel Bead Kit version 2 protocol. ScRNAseq libraries was obtained and RNA sequencing was performed that passed quality control (Novogene).