GSM5841324: Cardiac NCCs, WT; Mus musculus; RNA-Seq
E9.5 embryos were harvested and briefly placed ion ice-cold DPBS and then placed on a plastic petri dish resting on ice. Using insulin needles, the cardiac neural crest is dissected out from the mid otic placode to the posterior edge of the 4th somite. The tissues were removed and placed in a 1.5mL conical snap cap plastic tube on ice and 1x enzyme digestion buffer containing 100 μL of B. licheniformis stock (100mg/ml, Sigma P5380), 895 μL of DPBS and 5 μL of 0.5 M EDTA was added. The tube was quickly shaken thoroughly to mix and resuspend, incubated in ice water bath, and triturated using a cut-down 1ml pipet tip for 15s every 2 min. Cells were filtered through a 40 μm strainer, rinsed with 10 mL of ice-cold PBS/BSA, weighed and subsequently 300 g of the cells were transferred to 15 mL conical tube and centrifuged at 300X for 5 min at 4°C and the supernatant was discarded. The pelleted cells were then resuspended in 100 μL of ice-cold DPBS and counted on a Countess II (ThermoFisher Scientific). Cell count and viability was determined using trypan blue dye exclusion 10X Single Cell 3' v3 lib prep protocol ScRNA-Seq