GSM6127476: pilot tbx16-msgn1 snRNAseq, rep1; Danio rerio; RNA-Seq
Embryos were transferred, one by one, into separate wells of a 96-well V-bottom plate containing 75μL of 1X TrypLE + 2 mg/mL Collagenase P. Embryos were then dissociated by manual trituration at 30℃ once every 5 minutes. Dissociation continued until no visible chunks were present under a dissecting scope, which took between 20-40 minutes depending on embryo stage (e.g. 20 minutes for 18hpf and 40 minutes for 72hpf). Stop solution [1x dPBS, 5% FBS ] was then added to each well to quench the proteases. 3' end capture of polyAdenylated transcripts. In situ reverse transcription with barcoded RT primers followed by second strand synthesis, tagmentation and index PCR gives each nucleus and all the RNA molecules contained within it, a unique combination of barcode sequences. These barcodes are then demultiplexed to create single cells in silico.