|
| Status |
Public on Mar 30, 2011 |
| Title |
Tet1 KD ES cells, DNA methylation by MeDIP-chip (2/4) |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Tet1 KD ES cells, whole cell extract
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Mouse ES cells
strain: E14Tg2A
knockdown: Tet1
sample type: input DNA
|
| Growth protocol |
Mouse ES cells were maintained on gelatin-coated dishes in Glasgow Minimum Essential medium (GMEM; GIBCO), supplemented with 15% heat-inactivated fetal bovine serum, 55 mM b-mercaptoethanol (GIBCO), 2mM L-glutamine, 0.1mM MEM non-essential amino acid, 5,000 units/ml penicillin/streptomycin and 1,000 units/ml of ESGRO (Chemicon) under feeder-free conditions.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
MeDIP-chip was perfromed as previously described with minor modifications (Weber et al., 2005). Briefly, 5 ug sonicated, heat-denatured genomic DNA was immunoprecipitated with 5 ul of monoclonal antibody against 5-methylcytidine (Eurogentec) at 4 C overnight. Immunoprecipitated methylated DNA was collected with 30 ul Dynal protein-G beads for 2 h at 4 C. ChIP-chip assay was performed with about 10 million cells following Agilent mammalian ChIP-on-chip protocol (March, 2006). Briefly, cells were cross-linked with formaldehyde for 10 min at room temperature. Sonicated chromatin was immunoprecipitated overnight at 4 C with 10 ug immobilized (on 100 ul Dynal protein-G beads, Invitrogen) antibodies to Ezh2 (Active Motif, 39103).
|
| Label |
Cy3
|
| Label protocol |
Immunoprecipitated and input DNA were amplified using the Whole Genome Amplification Kit (Sigma), and 5 ug of amplified DNA were labeled (5’ Cy5- or Cy3-random nonamers, TriLink Biotechnologies) using the standard protocol (NimbleGen Arrays User’s Guide for ChIP-chip analysis).
|
| |
|
| Channel 2 |
| Source name |
Tet1 KD ES cells, immunoprecipitated DNA by 5-methylcytosine antibody
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Mouse ES cells
strain: E14Tg2A
knockdown: Tet1
sample type: immunoprecipitated DNA by 5-methylcytosine antibody
antibody: 5-methylcytosine antibody
antibody manufacturer: Eurogentec
antibody catalog #: BI-MECY_0100
|
| Growth protocol |
Mouse ES cells were maintained on gelatin-coated dishes in Glasgow Minimum Essential medium (GMEM; GIBCO), supplemented with 15% heat-inactivated fetal bovine serum, 55 mM b-mercaptoethanol (GIBCO), 2mM L-glutamine, 0.1mM MEM non-essential amino acid, 5,000 units/ml penicillin/streptomycin and 1,000 units/ml of ESGRO (Chemicon) under feeder-free conditions.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
MeDIP-chip was perfromed as previously described with minor modifications (Weber et al., 2005). Briefly, 5 ug sonicated, heat-denatured genomic DNA was immunoprecipitated with 5 ul of monoclonal antibody against 5-methylcytidine (Eurogentec) at 4 C overnight. Immunoprecipitated methylated DNA was collected with 30 ul Dynal protein-G beads for 2 h at 4 C. ChIP-chip assay was performed with about 10 million cells following Agilent mammalian ChIP-on-chip protocol (March, 2006). Briefly, cells were cross-linked with formaldehyde for 10 min at room temperature. Sonicated chromatin was immunoprecipitated overnight at 4 C with 10 ug immobilized (on 100 ul Dynal protein-G beads, Invitrogen) antibodies to Ezh2 (Active Motif, 39103).
|
| Label |
Cy5
|
| Label protocol |
Immunoprecipitated and input DNA were amplified using the Whole Genome Amplification Kit (Sigma), and 5 ug of amplified DNA were labeled (5’ Cy5- or Cy3-random nonamers, TriLink Biotechnologies) using the standard protocol (NimbleGen Arrays User’s Guide for ChIP-chip analysis).
|
| |
|
| |
| Hybridization protocol |
34 ug of Cy5- and Cy3-labeled samples were combined and hybridized to each mouse whole genome tiling microarray (Roche/NimbleGen 4-array set) for 16-20 h at 42 ˚C using NimbleGen hybridization System 4.
|
| Scan protocol |
Microarrays were scanned using the Agilent scanner at 5 micron resolution.
|
| Description |
IP/input
|
| Data processing |
Data were extracted and analyzed using NimbleScan v2.5 (Roche/NimbleGen). For identifying probes associated with a significant decrease in Ezh2 levels or a significant increase in DNA methylation level in Tet1 KD ES cells as compared to Con KD cells, a non-parametric one-sided Kolmogorov-Smirno (KS) test was used (KS score>1.3).
|
| |
|
| Submission date |
Jan 24, 2011 |
| Last update date |
Mar 30, 2011 |
| Contact name |
Hao Wu |
| E-mail(s) |
[email protected]
|
| Phone |
617-713-8660
|
| Organization name |
Harvard Medical School/HHMI
|
| Department |
Genetics
|
| Lab |
Yi Zhang
|
| Street address |
149G Warren Alpert Building, 200 Longwood Avenue
|
| City |
Boston |
| State/province |
Massachusetts |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL7524 |
| Series (2) |
| GSE26827 |
Dual functions of Tet1 in transcriptional regulation in mouse embryonic stem cells (ChIP-chip and MeDIP-chip) |
| GSE26833 |
Dual functions of Tet1 in transcriptional regulation in mouse embryonic stem cells |
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