title: XY_E6
organism: Danio rerio
description: Protocols: Around 100 Tg(Smarcd3-F6: EGFP) embryos were dechorionated with pronase (Sigma, Cat# 11459643001) and transferred to a 1.5mL Eppendorf tube when they reached bud stage. After incubated in 200ul calcium-free Ringer solution (116 mM NaCl, 2.6mM KCl, 5mM HEPE, pH 7.0) for 5 min, embryos were transferred into a 24-well plate filled with 500 ul TrypLE solution (GIBOCO, TrypLE Express Enzyme, cat #: 12604-013) for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single-cell suspension. The cell suspension was transferred into 200ul ice-cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice-cold DMEM with 10% FBS before resuspended in 500 ul ice-cold DMEM with 1% FBS. Right before the FACS, cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids-UHN Flow and Mass Cytometry Facility. Cell-doublets and dead cells were excluded based on forward scatter, side scatter and DAPI channel. GFP+ and GFP- cells were sorted into 100% FBS and subjected to RNA-seq or procedures immediately once sorting finished. 30,000-50,000 GFP+ cells and 100, 000 GFP- cells were usually collected in one sort. Single-cell cDNA libraries were prepared using Fluidgim C1 system. After FACS, GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400-500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription, PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30-40%, 96 single-cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit (Illumina, Cat# FC-131-1096).