title: GSM6670935: 7 dpa, scRNA-seq; Danio rerio; RNA-Seq
library_construction_protocol: Tissue from 20 adult zebrafish was pooled for each group. Regenerative caudal fin tissue was collected into 1x PBS on ice, diced, then washed with L-15 media supplemented with 1X GlutaMax, 1X Antibiotic-Antimycotic and 1X Penicillin-Streptomycin (all Thermo Fisher). The tissue was then enzymatically digested using 0.25% Trypsin-EDTA (Thermo Fisher) and Liberase DL (2 mg/ml, Sigma) at room temperature for 20 min followed by mechanical dissociation by pipet titration every 5 minutes. Enzymatic digestion was inhibited by the addition of 20% fetal bovine serum. Cells were filtered through a 100 μm cell strainer and then pelleted by centrifugation at 0.2 g for 2.5 minutes. The cell pellet was washed with PBS and re-suspended in PBS + 0.04% bovine serum albumin. Library was performed according to the manufacturer's instructions (single cell 3' v3 protocol, 10x Genomics). Briefly, cells from fin tissue 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, amplified by PCR, and the appropiate sized fragments were selected for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.