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 ATAC-seq procedures immediately once sorting finished. 30,000-50,000 GFP+ cells and 100, 000 GFP- cells were usually collected in one sort. 30, 000-50,000 cells obtained from FACS were used for nuclei prep. Nuclei for ATAC-seq experiments were purified following the published protocol (doi:10.1002/0471142727.mb1300s82). ATAC-seq libraries were generated using the published protocol (doi:10.1002/0471142727.mb1300s82) with minor modifications. After tagmentation reaction, transposed DNA fragments were amplified using the following PCR condition (1 cycle of 72 C for 5 min and 98 C for 30 s, followed by 12 cycles of 98 C for 10 s, 63 C for 30 s and 72 C for 1 min). Amplified libraries were purified twice with Agencourt Ampure XP beads (beckman coulter, Cat#A63880) with a bead-to-sample ratio of 1.8:1. Qubit fluorometer and Aglient Bioanalyzer were used to check library quality and concentration.
attributes.tag: ENA first public
attributes.value: 2018-08-21
attributes.tag: ENA last update
attributes.value: 2017-10-03
attributes.tag: External Id
attributes.value: SAMEA104327793
attributes.tag: INSDC center alias
attributes.value: Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto
attributes.tag: INSDC center name
attributes.value: Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto
attributes.tag: INSDC first public
attributes.value: 2018-08-21T17:02:16Z
attributes.tag: INSDC last update
attributes.value: 2017-10-03T08:58:29Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6078:WL441_10hpf_Smarcd3F6_danrer_FACS-GFPnega_ATAC_R2
attributes.tag: age
attributes.value: 10
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: zebrafish
attributes.tag: developmental stage
attributes.value: gastrula bud
attributes.tag: genotype
attributes.value: Smarcd3-F6:EGFPhsc70
attributes.tag: organism part
attributes.value: whole organism
attributes.tag: phenotype
attributes.value: GFP negative
attributes.tag: replicate
attributes.value: 2
attributes.tag: sample name
attributes.value: E-MTAB-6078:WL441_10hpf_Smarcd3F6_danrer_FACS-GFPnega_ATAC_R2