GSM2069431: EC-repair1; Danio rerio; RNA-Seq
Focused photoconversions of endothelial end cells or macrophages at three lesion stages (uninjured control, macrophage arrival, and macrophage traction) were achieved by irradiating Tg(flk1:DenNTR; coro1a:eGFP) or Tg(coro1a:Kaeda; flk1:eGFP) transgenic larvae with a 405-nm laser equipped on an LSM780NLO confocal microscope for 30 seconds. About eighty cells (repairing macrophages or endothelial end cells) in twenty animals were photoconverted. After photoconversion, zebrafish heads were dissected, collected, and homogenized in 1 ml 0.5% trypsin at 4 °C. The homogenized cell suspension was centrifuged at 1000x g at 4 °C for 5 minutes. Then, the supernatant was removed and the cell pellet was resuspended in PBS, followed by cell sorting by flow cytometry (Moflo XDP, Beckman). cDNA libraries were generated from these sorted cells using the Smart-seq2 protocol. RNA sequencing was performed using the PE100 strategy (HiSeq 2500, Illumina)