H1 hESCs were plated on Matrigel (BD Biosciences)-coated plates, and maintained in mTeSR (StemCell). Before purification, cells were trypsinized to single cells and TRA-1-60 expressing cells were isolated by using MACS cell separation columns (Miltenyi Biotec). Isolated cells were tested by flow cytometry, and samples with >99% purity were used. IMR90 human primary lung embryo fibroblasts (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) plus 10% FBS (Hyclone), 100 U/ml penicillin (Gibco), and 100 µg/ml streptomycin (Gibco) at 37°C in 5% CO2. Growing cells with 50~70% confluence were used for further analysis. Human primary hepatocytes (Zen-bio #HP-F) were grown in Hepatocyte Maintenance Medium (Zen-Bio #HM-2), and passage 4 was used. Human ESCs (HSF1) were differentiated to neural progenitor cells (NPCs) in DMEM:F12 (Gibco) plus B27 (Gibco), N2-supplement (Gibco), 20ng/ml bFGF (R and D systems), 1µM Retinoic Acid (Sigma), and 1µM Smoothened Agonist (Calbiochem). NPCs were mechanically isolated from culture based on rosette morphology as described(Elkabetz et al., 2008) and expanded in DMEM:F12 plus B27, N2-supplement, 20ng/ml bFGF, and 50ng/ml EGF (Gibco). NPCs were further differentiated to neurons and glia by withdrawal of the maintenance factors (bFGF and EGF) for 10 days. Human keratinocytes were cultured per manufacturer’s protocol in KSFM (Invitrogen). To induce differentiation, calcium chloride was added to 1.5mM for 48 hours(Lowry et al., 2005).
Extracted molecule
genomic DNA
Extraction protocol
Cells were fixed with 1% formaldehyde for 10 minutes at 37 degree followed by incubation with 140 mM glycine for 15 minutes at room temperature. Nuclear extracts were sonicated to get 150 ~ 500 bp genomic DNA fragments, diluted, precleared with 50 µl of Invitrogen Dynabeads Protein A (for Rabbit IgG) or Dynabeads Protein G (for mouse IgG) for 1 hour at 4 degree, and the supernatant was incubated with primary antibody overnight at 4 degree. Appropriate Dynabeads were then added to the solution and incubated for another 3 hours before washing steps. Reverse crosslinking of the precipitated complex was performed at 65 degree overnight followed by RNase A digestion, proteinase K treatement, phenol/chlorform extraction, and ethanol precipitation. DNA pelletes were air dried, and disolved in ddH2O.
Label
Cy3
Label protocol
DNA were labeled with Cy3- or Cy5-dCTP by using Invitrogen BioPrime DNA Labeling kit following manufacturere's instructions.
H1 hESCs were plated on Matrigel (BD Biosciences)-coated plates, and maintained in mTeSR (StemCell). Before purification, cells were trypsinized to single cells and TRA-1-60 expressing cells were isolated by using MACS cell separation columns (Miltenyi Biotec). Isolated cells were tested by flow cytometry, and samples with >99% purity were used. IMR90 human primary lung embryo fibroblasts (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) plus 10% FBS (Hyclone), 100 U/ml penicillin (Gibco), and 100 µg/ml streptomycin (Gibco) at 37°C in 5% CO2. Growing cells with 50~70% confluence were used for further analysis. Human primary hepatocytes (Zen-bio #HP-F) were grown in Hepatocyte Maintenance Medium (Zen-Bio #HM-2), and passage 4 was used. Human ESCs (HSF1) were differentiated to neural progenitor cells (NPCs) in DMEM:F12 (Gibco) plus B27 (Gibco), N2-supplement (Gibco), 20ng/ml bFGF (R and D systems), 1µM Retinoic Acid (Sigma), and 1µM Smoothened Agonist (Calbiochem). NPCs were mechanically isolated from culture based on rosette morphology as described(Elkabetz et al., 2008) and expanded in DMEM:F12 plus B27, N2-supplement, 20ng/ml bFGF, and 50ng/ml EGF (Gibco). NPCs were further differentiated to neurons and glia by withdrawal of the maintenance factors (bFGF and EGF) for 10 days. Human keratinocytes were cultured per manufacturer’s protocol in KSFM (Invitrogen). To induce differentiation, calcium chloride was added to 1.5mM for 48 hours(Lowry et al., 2005).
Extracted molecule
genomic DNA
Extraction protocol
Cells were fixed with 1% formaldehyde for 10 minutes at 37 degree followed by incubation with 140 mM glycine for 15 minutes at room temperature. Nuclear extracts were sonicated to get 150 ~ 500 bp genomic DNA fragments, diluted, precleared with 50 µl of Invitrogen Dynabeads Protein A (for Rabbit IgG) or Dynabeads Protein G (for mouse IgG) for 1 hour at 4 degree, and the supernatant was incubated with primary antibody overnight at 4 degree. Appropriate Dynabeads were then added to the solution and incubated for another 3 hours before washing steps. Reverse crosslinking of the precipitated complex was performed at 65 degree overnight followed by RNase A digestion, proteinase K treatement, phenol/chlorform extraction, and ethanol precipitation. DNA pelletes were air dried, and disolved in ddH2O.
Label
Cy5
Label protocol
DNA were labeled with Cy3- or Cy5-dCTP by using Invitrogen BioPrime DNA Labeling kit following manufacturere's instructions.
Hybridization protocol
Labeled DNA were mixed with human Cot-1 DNA, Agilent aCGH Blocking solution, and Agilent aCGH hybridization buffer. After 5-minute denaturing, and 30-minute incubation at 37 degree, mixed solution was added to array, and hybridized for 40 hours at 65 degree.
Scan protocol
Scanned on Agilent G2505C scanner. Images were quantified using Agilent Feature Extraction 10.5.1.1
Description
SIRT1 knockdown
Data processing
Agilent ChIP Analytics was used for inter-array median normalization. Background subtraction, common features median normalization, and dye bias normalization were performed by using MATLAB.
