GSM9136973: Pogona vitticeps dorsal skin cells, embryonic day 37; Pogona vitticeps; RNA-Seq
Dorsal skin from corn snake and bearded dragon embryos was dissected from either individual or pooled embryos. Tissues were collected in ice-cold Hank's Balanced Salt Solution (HBSS; Sigma, M4780). Late-stage samples (harvested between E47 and E54) were incubated overnight at 4 °C in DMEM (ThermoFisher, 11960044) supplemented with 0.25% Dispase II (Sigma, D4693) and 10 mM HEPES pH 8 (Fisher, BP310) prior to enzymatic digestion. All tissues were then incubated in 1 mL of digestion solution containing 2.5 mg/mL Collagenase Type II (Worthington, LS004176), 2 mM CaCl₂ (Sigma, 21110), and 1 U/μL DNase I (Worthington, LS002139) in HBSS. For bearded dragon samples older than E33, the digestion solution was further supplemented with 1 U/μL Hyaluronidase (Worthington, LS002592). Samples were digested on a rotating wheel at 35 °C for 35 minutes. Following digestion, samples were diluted in 4 mL of HBSS containing 0.25% BSA (Sigma, A3294), centrifuged at 300 × g for 10 minutes, and the resulting cell pellets were resuspended in 5 mL of 0.25% BSA in HBSS. The suspension was filtered through 40 μm filters (Corning, 352340), centrifuged again at 300 × g for 10 minutes, and resuspended in 110 μL of 0.25% BSA in HBSS. A 10 μL aliquot was taken for microscopy, and the remaining cell suspension was cryopreserved at –80 °C in a freezing medium consisting of DMEM (ThermoFisher, 11960044) with 10% fetal calf serum and 10% DMSO (Sigma, D2653) using a controlled-rate cooling box. Thawed cell suspensions were counted, and single-cell RNA-seq libraries were generated using the 10X Genomics Chromium Single Cell 3′ v3 kit. Libraries were sequenced on an Illumina NovaSeq 6000 platform, targeting 5,000 cells and 50,000 reads per cell, resulting in approximately 250 million reads per sample.