GSM7316667: Wildtype 30 hours post-fertilization Zebrafish Embryo - Replicate 2; Danio rerio; RNA-Seq
Wildtype zebrafish embryos, expressing the Tg(nkx3.1:Gal4;UAS:NTR-mCherry)ca101 transgene, were dissociated, assessed for viability, processed, resuspended in a designated solution, and subsequently sorted via fluorescence-activated cell sorting to isolate red fluorescent nkx3.1-transgene expressing cells. The sample was prepared according to 10X Genomics ChromiumTM Single Cell 3' Reagent Guidelines v3 Chemistry. Briefly, single cells were sorted into 0.1% BSA–HBSS and partitioned into Gel Bead-In-EMulsions (GEMs) using 10xTM GemCodeTM Technology. This process lysed cells and enabled barcoded reverse transcription of RNA, generating full-length cDNA from poly-adenylated mRNA. DynaBeads® MyOneTM Silane magnetic beads were used to remove leftover biochemical reagents, then cDNA was amplified by PCR over 10 cycles. Quality control size gating was used to select cDNA amplicon size prior to library construction. Read 1 primer sequences were added to cDNA during GEM incubation. P5 primers, P7 primers, i7 sample index, and Read 2 primer sequences were added during library construction. Quality control and cDNA quantification was performed using Agilent High Sensitivity DNA Kit. Sequencing was performed using Illumina NovaSeq S2 flow cell at the Center for Health Genomics and Informatics at the University of Calgary.