GSM9553343: Embryo, Hpf24, Genotyping-only, rep1; Strongylocentrotus purpuratus; RNA-Seq
Staged embryos were pelleted at 150g at 4°C for 5 minutes, washed with calcium-free artificial sea water (CFSW) [454mM NaCl, 9mM KCl, 48mM MgSO4, 6mM NaHCO3; pH adjusted to 8.2], and incubated in hyaline extraction medium (HEM) supplemented with BSA [NaCl 300mM, KCl 10mM, MgSO4 10mM, Glycine 300mM, Tris 10mM, EGTA 2mM; pH adjusted to 8.2; supplemented with BSA 2mg/mL] on ice for 5 minutes. Embryos were dissociated by gentle trituration with a 25ml serological pipet until reaching >95% single cells. Dissociated cells were then washed twice in CFSW and remaining clumps of cells were filtered out using a 40μm cell strainer. Cell viability was assessed using propidium iodine (Sigma Aldrich) staining. Only samples with > 90% viability were processed further. Cells were then diluted to 5x106 cells/ml in CFSW and fixed by adding ice-cold MeOH (to 90%) dropwise while gently vortexing and stored at -20°C until processing. Fixed cells were rehydrated prior to library preparation. Therefore, cells were spun down (1000g, 5min at 4°C) and hydrated by two washes in CFSW with 0.1% BSA and 1x SUPERaseIn RNase inhibitor (both Fisher scientific). Rehydrated cells were diluted in Hanks balances salt solution (HBSS) with 0.04% BSA, followed by library preparation according to manufacturer's instructions (10x Genomics). For scSLAM-seq, single cells were incubated with 10mM iodoacetamide overnight in 80% MeOH in CFSW at room temperature with gentle rotation and rehydrated in presence of 100mM DTT.