GSM5014336: Zsphere-scRNA; Danio rerio; RNA-Seq
At high stage, embryos were transferred in agar coated 6 cm petri dish and manually dechorionated in embryo media. At sphere stage, the embryo media was sequentially replaced with Dulbecco Modified Eagle's Medium (DMEM F-12) with 0.04% BSA and the blastula cups were dissected away from 25 embryos and transferred to an Eppendorf tube containing 100µl of DMEM-F12 +0.04% BSA. The cell suspension was pelleted at 400 rcf for 30sec. The media was then removed and cells were suspended in 200µl of ice-cold PBS+0.04% BSA to which 800µl of MeOH were added dropwise. Samples were kept in ice for 15 minutes and finally transferred to -20°C until use. For rehydration samples were kept in ice for 15 min and cells were pelleted at 1000rcf for 5 min at 4°C. Cells were resuspended in 100µl ice cold PBS + 0.04% BSA and the cell suspension was loaded into 10X Genomics Single Cell 3′ Chip The quality and concentration of the cell suspension was measured using an automatic cell counter. For sequencing, the ample was diluted to a concentration of ~1000 cells/ul and approximately 10 000 cells were loaded into a Chromium Chip for use in the 10X Chromium Controller. The library was sequenced on a HiSeq 4000. 10x Chromium Single Cell 3'