Song:Experimental Design For Brainbow: Difference between revisions
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<b>Aim 1.</b>To generate cell lines random-integrated with brainbow cassette | <b>Aim 1.</b> To generate cell lines random-integrated with brainbow cassette | ||
*To construct lentiviral vector carrying Brainbow-2.1 cassette | *To construct lentiviral vector carrying Brainbow-2.1 cassette | ||
*Lentivirus package and transfection | *Lentivirus package and transfection | ||
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<b>Aim 2.</b>To introduce inducible Cre into brainbow-integrated cell lines | <b>Aim 2.</b> To introduce inducible Cre into brainbow-integrated cell lines | ||
*To construct inducible Cre with a drug-selection marker | *To construct inducible Cre with a drug-selection marker | ||
**CAG-CreER regulated by tamoxifen | **CAG-CreER regulated by tamoxifen | ||
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<b>Aim 3.</b>To establish live cell imaging and automated lineage tracking system | <b>Aim 3.</b> To establish live cell imaging and automated lineage tracking system | ||
*Acquiring fluorescence images from all channel at a defined interval | *Acquiring fluorescence images from all channel at a defined interval | ||
*Processing acquired images and segmenting cells from the images | *Processing acquired images and segmenting cells from the images | ||
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<b>Aim 4.</b>To manually isolate single cell from differentiation culture | <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 | *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 single cells by local trypsination and micromanipulation | ||
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<b>Aim 5.</b>To perform single-cell transcriptomic and epigenomic analysis | <b>Aim 5.</b> To perform single-cell transcriptomic and epigenomic analysis | ||
*Single cell toto-RNAseq | *Single cell toto-RNAseq | ||
*Characterizing DNA accessibility on limited cells by THS-seq | *Characterizing DNA accessibility on limited cells by THS-seq |
Latest revision as of 19:16, 6 September 2013
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