GSM9771929: Killifish incipient aggregate stage (72 hpf); Nothobranchius furzeri; RNA-Seq
Embryos were manually dechorionated with forceps in ice cold DMEM/F12 (Gibco, 21-041-025) and transferred to lo-bind 1.5 mL Eppendorf tubes on ice. Cells were spun down at 500xg for 3 min at 4°C. Cells were dissociated in Accutase (Innovative Cell Technologies, AT104) at 37°C for 7-20 min depending on embryo stage (older embryos required longer dissociation). Percent viability was determined by counting DAPI+, Calcein-AM+ (Invitrogen, C1430) live cells and DAPI+, Calcein-AM- dead cells on a disposable hemocytometer (Bulldog Bio, DHC-N420). 40,000 cells were loaded onto the V4 T20 PIP-seq kit (formerly Fluent Bio, now the Illumina Single Cell 3'RNA Prep Kit) for each timepoint except for the 115hpf timepoint which consisted of a 1:1 mix of killifish and stage-matched zebrafish cells used to initially assess the efficacy of the PIP-seq kit which, at that time, had not yet been used on teleosts. Followed V4 P20 PIPseq protocol. cDNA fragment analysis of scRNAseq libraries was performed using the 4200 TapeStation System. Libraries were sequenced with a NextSeq 2000 P3 100 cycle kit (52hpf, 72hpf, 96hpf) or NovaSeq 6000 S4 300 cycle kit (115hpf, a gift from Fluent Biosciences) using the following configuration: read 1: 54, i7 index: 8, i5 index: 8, read 2: 68.