GSM6817224: Donor #3 Fibrosa; Homo sapiens; RNA-Seq
Briefly, 500 units/mL of collagenase type I (MP Biomedical), 500 units/mL of collagenase type II (MP Biomedical), 150 units/mL collagenase Type XI (Sigma-Aldrich), 60 units/mL hyaluronase type Is (Sigma-Aldrich), and 60 units/mL DNASE I (Zymo) were dissolved in HBSS without calcium and magnesium and filtered through a 0.45 um syringe filter. One and a half AV leaflets were placed in a 35 mm dish with the desired layer for isolation face-up, then 2 mL of dissociation buffer was placed on the upwards facing layer and incubated for 45 min at 37 ºC. After incubation, cells from the layer in contact with the dissociation buffer were gently scraped off using a sterile cotton swab, blocked with 1 mL of fetal bovine serum (FBS) and passed through a 70 μm cell strainer. The suspension was centrifuged for 5 min at 500 x g and resuspended in > 50 μL of 2% BSA in PBS at a concentration of 1000 cells/μL. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics).