GSM4681962: Cornea Stage 49-51; Xenopus laevis; RNA-Seq
A total of approximately 80 corneas in three separate glass depression dishes containing 400 l 1X DPBS, on ice. Care was taken to ensure that corneas were carefully removed from the tip of the forceps and that they remained submerged in the 1X DPBS solution. Using a 200 l pipette tip, most of the DPBS was gently removed without disturbing the corneas at the bottom of each glass dish. To dissociate the corneal epithelial tissue, ~350 l of Accutase (CellnTec Advanced Cell Systems, Bern, Switzerland) was added to each dish, and the dishes were covered with plastic petri dish lids (35 X 10 mm, Corning, 351008) flat side down, and wrapped tightly with lab tape to secure them and prevent desiccation. Next, the dishes were incubated in an pre-hyb oven at 37C for 35 minutes with intermittent trituration using a 200 l pipette tip. To stop the reaction, 200 l of media containing 61% L-15 (Invitrogen, Carlsbad, CA), 0.5 M EDTA (Invitrogen, Carlsbad, CA) and 10% Fetal Bovine Serum (Invitrogen, Carlsbad, CA) was added to each dish. For mechanical dissociation the suspension was finally triturated 8-10 times using the 200 l pipette tip. The three cell suspensions were pooled in a 2ml LoBind microcentrifuge tube (Eppendorf, 022431048), spun at 325g for 5 minutes in a fixed angle centrifuge, and resuspended in 200 l L-15 with 10% FBS and 0.5 M EDTA. Finally, the cells were passed through a 40 m FlowMi cell strainer (Bel-Art, Wayne, NJ; Cat 136800045) and collected in 2ml LoBind tube. Cell viability was assessed using a dual-fluorescence test by staining the cells with acridine orange (AO) and propidium iodide (PI) The single cell RNA-seq library was generated using the 10X Genomics Chromium Single Cell 3' Kit v3 (Location, Address) and sequenced with Illumina NovaSeq 6000 on a SP flow cell to obtain 150bp paired reads.
GEO Accession
GSM4681962