GSM7706846: mitfa_low, zf, scRNAseq; Danio rerio; RNA-Seq
Tg(mitfa:GFP) zebrafish embryos at 24hpf were dechorinated using 5 mg/ml pronase for 10-15 min, deyolked in Ringer's solution (ice cold) using a micropipette tip and spun at 100 g for 2 min at 4°C . The embryo bodies were trypsinized using TrypLE Express for 15 or 30 min at room temperature, after discarding the supernatent. Clumps were removed by passing the cells through a 70 μm cell strainer and washing twice with ice cold phosphate-buffered saline. The cells expressing low and high levels of mitfa:GFP were then sorted using a flow cytometer and taken for library preparation. Libraries was prepared according to the manufacter's instructions (single cell 3' v3.1 (single index) protocol, 10x Genomics). Briefly, 24hpf mitfa:GFP low and high zebrafish cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and sample index were added.