GSM9282612: Basal epidermal cells, 4 dpa; Danio rerio; RNA-Seq
Dissociated cells from zebrafish caudal fin were prepared by the method of Thompson et al. 2020(47). Regenerating caudal fin samples were collected at time points ranging from 1 to 4 dpa via excision of the distal most 2 mm with a razor blade. For uninjured controls (0 dpa), a 2 mm section of mid caudal fin was excised that corresponded to the sampled area in injured fish. Cells were dissociated with 1.18 U/ml Liberase DH (Roche) in Hank's Balanced Salt Solution (Gibco) with stirring at 37 °C for 2 h, collected in 15 min increments, then strained through a 40 μm nylon cell strainer (Corning). Cell suspensions were stained with SYTOX Blue (Invitrogen) and sorted for exclusion of the dye via FACS (Astrios Cell Sorter via Duke Cancer Institute Flow Cytometry Core Facility). In experiments examining basal epidermal cell populations, cells were further sorted by endogenous red fluorescence of the krt1‑19e:h2az2a‑mCherry transgene and exclusion of green fluorescence from the krt4:LY‑EGFP transgene, which mark basal epidermal and superficial epidermal populations, respectively. FACS-sorted suspensions were then resuspended in PBS with 400 μg/mL non-acetylated bovine serum albumin (Sigma). An aliquot of the suspension was counted by hemacytometer and checked for viability with 0.4% trypan blue (Sigma), which ranged from 60-98% between samples. Cells were encapsulated in gel bead emulsions and barcoded using the Chromium Controller (10x Genomics) with target yields of 6,000-10,000 cells. Preparation of cDNA and Illumina sequencing libraries was carried out according to the Chromium Single Cell 3' Reagents Kit v.2 (10x Genomics).