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