The scRNA-Seq libraries were prepared following the published protocol with minor modifications (11 (link)). Briefly, cells undergoing reprogramming were trypsinized into single cells at each time point. Cells were washed three times with C1 DNA-seq Cell Wash Buffer (Fluidigm). Cells at a concentration of 300 to 600 cells/μl were mixed with C1 Cell Suspension Reagent at a ratio of 3:2. Next, the cell mixture was loaded into C1 Single-Cell Auto Prep IFC (integrated fluidic circuits) microfluidic chips according to the “STRT seq, 1862× (10-17 μm diameter cells)” protocol. Cells were captured at the 96 capture sites in the microfluidic chip and stained using a green fluorescent calcein-AM dye (LIVE/DEAD cell viability assay, Life Technologies) and imaged by a Leica CTR 6000 microscope. mRNA-Seq libraries were prepared following the Generate complementary DNA (cDNA) libraries with the C1 Single-Cell mRNA Seq HT IFC and Reagent Kit V2 manual.
Protocol detail
Single-cell RNA-sequencing of reprogramming cells
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Assay
Buffer
Calcein
Cdna
Cell
Cell viability
Chip
Chips seq
Microscope
Mrna
Scrna seq
Corresponding Organization :
Other organizations : Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Nanyang Technological University, National University of Singapore, Singapore Bioimaging Consortium, Pohang University of Science and Technology, Duke-NUS Medical School, Institute of Bioengineering and Nanotechnology, Institute for Basic Science, Radboud University Nijmegen, Massachusetts Institute of Technology, Broad Institute, Harvard University, Dana-Farber Cancer Institute, Boston Children's Hospital, Harvard Stem Cell Institute, Dana-Farber/Boston Children's Cancer and Blood Disorders Center, Mayo Clinic
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Variable analysis
independent variables
- Time point of cell reprogramming
- ScRNA-Seq library preparation
- Capture of cells in microfluidic chip
- Staining of cells with calcein-AM dye
- Imaging of cells
- MRNA-Seq library preparation
- Cell concentration (300 to 600 cells/μl)
- Cell washing with C1 DNA-seq Cell Wash Buffer
- Mixing of cell suspension with C1 Cell Suspension Reagent (3:2 ratio)
- Use of C1 Single-Cell Auto Prep IFC microfluidic chips
- Use of C1 Single-Cell mRNA Seq HT IFC and Reagent Kit V2
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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