GSM8171902: D51S_SAE_Fresh; Homo sapiens; RNA-Seq
LAE, SAE, and PCLS tissues were cryopreserved in CryoStor freezing medium after 0 (fresh), 2, and 14 days in culture. Cryovials were thawed, rinsed in PBS, placed into a 15 mL tube containing 5 mL of Accutase solution (Sigma-Aldrich, #A6964-100ML) with EDTA (invitrogen, #15575-038) (5 mM) and pronase (Roche, #10165921001) (1 mg/mL), and incubated on a shaker for 30 min at 37°C. Following incubation, the tube was centrifuged (600 g, 2 min, 4 oC), the supernatant was discarded, and 5 mL of Hank's Balanced Salt Solution (Ca-, Mg-) (gibco) buffer containing liberase (Roche, #05401119001) (50 mg/mL) and DNase (Roche, #10104159001) (25 ug/mL) was added to the tube. The sample was then incubated on the shaker for 10 min at 37°C. After incubation, the cell suspension was gently pipetted 20 times using a wide-orifice pipette tip. To neutralize enzymes, 500 mL of fetal bovine serum was added to the cell suspension, and the neutralized mixture was filtered through a 100 um filter, followed by centrifugation (600 g, 2 min, 4 oC). After removing supernatant, dissociated cells were resuspended in PBS (Ca-, Mg-) containing 0.01% Ultrapure BSA (Invitrogen, #AM2616), filtered through a 40-μm cell strainer (Bel-Art, H13680-0040), and centrifuged (600 g, 2 min, 4 oC). Cell count and viability was assessed using trypan blue dye exclusion with a Countess 3 automated cell counter (Thermo Fisher Scientific), and the cell concentration was adjusted to approximately 1000 cells/uL. The dissociated cells were used for 10X Genomics scRNA-seq library preparation. Single-cell suspensions targeting approximately 6,000 cells were loaded onto a 10x Genomics Chromium Next GEM Chip G (10x Genomics) device following the manufacturer's instructions. After GEM generation, the library preparation was performed following the instructions for the 10x Chromium Next GEM Single Cell 3' GEM Library kit v3.1 (10x Genomics).