Song:Experimental Design For Brainbow
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Aim 1. 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
Aim 2. 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
Aim 3. 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
Aim 4. 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
Aim 5. To perform single-cell transcriptomic and epigenomic analysis
- Single cell toto-RNAseq
- Characterizing DNA accessibility on limited cells by THS-seq