title: GSM6940304: 22-25 mm Adult Calvaria, ecadYFPdepleted, DAPI neg; Danio rerio; RNA-Seq
library_name: GSM6940304
library_construction_protocol: We dissected the calvaria from 20 juvenile (9-11mm) from ecad:YFP (one dataset) or sp7:GFP (one dataset), and separately 10 adult (22-25 mm) ecad:YFP (one dataset) or Tubingen (one dataset), fish away from brain tissue in Ringer's solution. 9-11 mm stages Calvaria were mechanically minced with a razor and all tissue was transferred to a 1.5 mL tube for enzymatic dissociation (0.25% trypsin (Life Technologies, 15090-046), 1 mM EDTA, and 400 mg/mL Collagenase D (Sigma, 11088882001) in PBS). Tissue was incubated on a nutator at 28.5 °C for ~45 min and the enzymatic reaction was stopped by adding 6X stop solution (6 mM CaCl2 and 30% fetal bovine serum (FBS) in PBS).  Cells were pelleted by centrifugation (300 g) at 4 °C, rinsed in suspension media (1% FBS, 0.8 mM CaCl2 (Sigma-Aldrich, St. Louis, MO) in phenol red-free Leibovitz's L15 medium (Life Technologies)), and resuspended in a final volume of 500 uL. DAPI was added to the resuspended cells, and cells were fluorescence-activated cell sorted (FACS) to isolate live cells that were DAPI negative and when applicative, ecad:YFP/sp7:GFP negative to deplete epithelial cells. scRNAseq libraries were prepared using 10X Genomics Chromium Single Cell 3′ Library and Gel Bead Kit v.2 according to the manufacturer's instructions and sequenced using Illumina NextSeq or HiSeq machines at a depth of at least 50,000 reads per cell for each library. Juvenile and adult stages were performed in replicates, and sequenced data was aligned using Cellranger v3.0.0 from 10X Genomics against the GRCz11 genome. All parameters were set to their default values.