GSM9230028: 48hpa-run2; Nematostella vectensis; RNA-Seq
Samples were washed with ice cold Calcium Magnesium Free (CMF) seawater briefly and the samples were allowed to settle. The CMF was then removed and they were resuspended in ACME (ACetic-MEthanol) prepared as described previously (Garcia-Castro et al. 2021) and tubes were left on the rocker for 20 min. The samples were then dissociated using a cut pipette tip every 5 min during the 20 min window and their dissociation status was checked until sufficient. Tubes were then spun at 400 rcf for 4 min at 4℃ and the supernatant was carefully removed without disturbing the loose cell pellet. The pellet was then resuspended with 1xPBS + 0.5% BSA + RNaseOUT (40U/ml) and DMSO was added to be 10% of the final volume. This cell mix was then stored at -80℃ until the day of  library preparation. On library preperation day, the tubes were left to thaw at RT and then spun again at 400 rcf for 4 min at 4℃ after which the cell pellet was resuspended in 1xPBS + 0.5% BSA + RNaseOUT (40U/ml) and the viability and concentration were then quantified using ViaStain™AOPI staining solution (Nexcelom) and the Cellometer X2. Libraries for sequencing were prepared using the standard 10X pipeline for scRNAsequencing