GSM9199074: Wound, 4 dpw; Danio rerio; RNA-Seq
Unwounded skin tissue from three adult zebrafish was collected by scraping off the skin and pooling in PBS (Mg2+ and Ca2+-free). In addition, the wound tissues for 2 dpw, 4 dpw and 6 dpw were collected using a biopsy punch with a diameter of 4 mm from three adult zebrafish each. PBS was replaced with 0.25 mg/ml Liberase TM in PBS solution and the tissues were incubated at 30oC for an hour with slow agitation (~300 rpm). The solution was pipetted every 10-15 minutes. After successful digestion of the tissue, the solution was mixed with 5% FBS/PBS and filtered through a cell strainer with 70 μm nylon mesh and then a cell strainer with 40 μm nylon mesh in 50 ml Falcon tubes. The falcon tubes were centrifuged at 500 x g for 5 minutes and the pellet was resuspended with 2% FBS/PBS. The viability of the cells were more than ninety percent. The single cell suspensions were kept on ice until they were processed at the Cologne Center for Genomics, University of Cologne. 10.000 cells per condition was aimed for single cell RNA sequencing. Single cell suspensions were processed, sequenced and single cell library construction was mapped to the zebrafish transcriptome by Cologne Center for Genomics, University of Cologne using 10x Genomics 3'scRNA seq application (Version 3'v.3.1) with Cell Ranger software (cellranger-6.1.2) used to generate feature-barcode matrices.