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Song:Experimental Design For Brainbow
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<b>Aim 1.</b> To generate cell lines random-integrated with brainbow cassette *To construct lentiviral vector carrying Brainbow-2.1 cassette *Lentivirus package and transfection *Isolating GFP-positive cells by flow cytometric cell sorting *Establishing integrated clonal cell line from single cell *Validating the persistency and efficiency of GFP expression after passaging and during differentiation <b>Aim 2.</b> To introduce inducible Cre into brainbow-integrated cell lines *To construct inducible Cre with a drug-selection marker **CAG-CreER regulated by tamoxifen **Or tetO-Cre regulated by doxycycline *Establishing Cre-integrated stable cell lines by drug selecting *Validating stochastic fluorescence labeling after the addition of tamoxifen or doxycycline to the culture <b>Aim 3.</b> To establish live cell imaging and automated lineage tracking system *Acquiring fluorescence images from all channel at a defined interval *Processing acquired images and segmenting cells from the images *To develop algorithms for automated lineage tracking <b>Aim 4.</b> To manually isolate single cell from differentiation culture *Determining the time required for XFP expression and for cells to complete mitosis after drug addition *Isolating single cells by local trypsination and micromanipulation *Isolating the differentiation derivatives of the siblings of the above step *Recording the spatial and color information of each isolated cells <b>Aim 5.</b> To perform single-cell transcriptomic and epigenomic analysis *Single cell toto-RNAseq *Characterizing DNA accessibility on limited cells by THS-seq
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