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Sample GSM661343 Query DataSets for GSM661343
Status Public on Sep 18, 2012
Title H3K9me2 in H1.5 knockdown IMR90 cells IP vs INPUT slide1
Sample type genomic
 
Channel 1
Source name human fetal lung fibroblasts
Organism Homo sapiens
Characteristics antibody: none
cell type: human fetal lung fibroblasts
cell line: IMR90
Growth protocol 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.
 
Channel 2
Source name human fetal lung fibroblasts
Organism Homo sapiens
Characteristics antibody: H3K9me2 antibody
cell type: human fetal lung fibroblasts
cell line: IMR90
antibody manufacturer: Millipore
antibody catalog #: 07-441
Growth protocol 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 H1.5 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.
 
Submission date Jan 25, 2011
Last update date Sep 18, 2012
Contact name Jing-Yu Li
E-mail(s) jingyuli@mednet.ucla.edu
Organization name University of California, Los Angeles
Street address 615 Charles E. Young Dr. South BSRB 357
City Los Angeles
ZIP/Postal code 90095
Country USA
 
Platform ID GPL4124
Series (2)
GSE26861 Genomic Binding of Human Linker Histone H1.5 during differentiation (ChIP-chip)
GSE26979 Human Linker Histone H1.5

Data table header descriptions
ID_REF
VALUE normalized log10 ratio Cy5/Cy3

Data table
ID_REF VALUE
1 -1.733448087e-001
2 0.000000000e+000
3 0.000000000e+000
4 -9.716471862e-001
5 6.209820683e-001
6 3.698117605e-001
7 -7.378084084e-002
8 -1.105002210e-001
9 -1.399109112e-001
10 -1.774161823e-001
11 8.842991869e-002
12 -5.601742806e-001
13 3.177621816e-001
14 0.000000000e+000
15 -2.630318611e-001
16 -6.799314555e-001
17 1.198980611e-001
18 0.000000000e+000
19 -3.222319694e-001
20 5.923669400e-002

Total number of rows: 243494

Table truncated, full table size 5714 Kbytes.




Supplementary file Size Download File type/resource
GSM661343_US82903519_251470612998_S01.txt.gz 70.1 Mb (ftp)(http) TXT
Processed data included within Sample table
Processed data are available on Series record

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