GSM4855811: Saline injected 5 day old embryo cells; Danio rerio; RNA-Seq
The embryos were euthanized with tricaine and ice added to egg water. The embryos were then added to methylcellulose and the heads of the embryos were removed using a straight blade sterile scalpel. The remaining portion of the embryos were collected and washed 2x with cold PBS at 700g for 5 min then dissociated with Liberase TL (Sigma) containing DNAse I (Sigma) in DMEM using gentleMACS (Miltenyi Biotec) in C tubes then cells were incubated at 370C for 10 minutes with continuous monitoring. When cell homogenate started to disappear, DMEM containing 10% FBS was added to stop the dissociation reaction. Cells were then filtered on 70µm filter and centrifuged at 1500 rpm for 10 min. Cells were counted on hemocytometer then dead cells were removed using dead cell removal microbead (Miltenyi Biotec) with MS column. Live cells were then centrifuged and resuspended in DPBS (without Ca2+ or Mg2+) and processed for single cell RNA sequencing on 10x genomics platform. Library preparation was performed usingChromium™ Next GEM Single Cell 3ʹ GEM, Library & Gel Bead Kit v3.1.