|
| Status |
Public on Jan 01, 2017 |
| Title |
ESC H3K4me1-Rep1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
H3K4me1 ChIP DNA from 46c ESC
|
| Organism |
Mus musculus |
| Characteristics |
cell type: 46c ESCs
chip antibody: H3K4me1
|
| Growth protocol |
GMEM+ Supplement
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Nuclei from 3x10^6 mESCs or sorted Sox1+ve NPCs were prepared and resuspended in NB-R (85 mM NaCl, 5.5% Sucrose, 10mM TrisHCl pH 7.5, 3 mM MgCl2, 1.5 mM CaCl2, 0.2 mM PMSF, 1 mM DTT), Micrococcal nuclease (MNase) digestion and native ChIP were performed as previously described (Eskeland et al. 2010, Pradeepa et al.2012) Antibodies used for ChIP were; H3K4me1 (Abcam ab8895) and H3K27ac (Millipore 07-360).
|
| Label |
Cy5
|
| Label protocol |
Ten nanograms (optimal) of input or ChIP DNA were amplified using the WGA2 whole genome amplification kit (Sigma). Amplified material was labelled with Cy3 or Cy5 by random priming according to the NimbleGen ChIP-chip protocol (Roche). In total, 2 or 3 biological replicates with dye swaps were hybridized for 20 h and washed according to manufacturer´s protocol. A custom 3x720K mouse tiling array (NimbleGen, Roche) containing 179,493 unique probes from different genomic regions, with each probe represented by 4 replicates was used.
|
| |
|
| Channel 2 |
| Source name |
Input DNA from 46c ESCs
|
| Organism |
Mus musculus |
| Characteristics |
cell type: 46c ESCs
control type: input DNA
|
| Growth protocol |
GMEM+ Supplement
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Nuclei from 3x10^6 mESCs or sorted Sox1+ve NPCs were prepared and resuspended in NB-R (85 mM NaCl, 5.5% Sucrose, 10mM TrisHCl pH 7.5, 3 mM MgCl2, 1.5 mM CaCl2, 0.2 mM PMSF, 1 mM DTT), Micrococcal nuclease (MNase) digestion and native ChIP were performed as previously described (Eskeland et al. 2010, Pradeepa et al.2012) Antibodies used for ChIP were; H3K4me1 (Abcam ab8895) and H3K27ac (Millipore 07-360).
|
| Label |
Cy3
|
| Label protocol |
Ten nanograms (optimal) of input or ChIP DNA were amplified using the WGA2 whole genome amplification kit (Sigma). Amplified material was labelled with Cy3 or Cy5 by random priming according to the NimbleGen ChIP-chip protocol (Roche). In total, 2 or 3 biological replicates with dye swaps were hybridized for 20 h and washed according to manufacturer´s protocol. A custom 3x720K mouse tiling array (NimbleGen, Roche) containing 179,493 unique probes from different genomic regions, with each probe represented by 4 replicates was used.
|
| |
|
| |
| Hybridization protocol |
The labeled ChIP DNA was hybridized for 16-18 h at 42C, and then washed using manufacturer's protocols (http://www.nimblegen.com/products/lit/lit.html)
|
| Scan protocol |
Arrays were scanned on a NimbleGen MS 200 Microarray scanner (Roche) using 100% laser power and 2μm resolution.
|
| Description |
ChIP-chip ESCs H3K4me1
|
| Data processing |
Raw signal intensities were quantified from TIFF images using MS 200 Data Collection software. Microarray data were analysed in R using the bioconductor packages Beadarray and Limma according to the Epigenesys NimbleGen ChIP-on-chip protocol 43. Replicate probe values were collapsed to the arithmetic mean prior to generating log2 (ChIP/Input) ratios.
|
| |
|
| Submission date |
Jul 07, 2016 |
| Last update date |
Jan 01, 2017 |
| Contact name |
Rob S Illingworth |
| E-mail(s) |
robert.illingworth@ed.ac.uk
|
| Phone |
01316519640
|
| Organization name |
The University of Edinburgh
|
| Department |
Centre for regenerative Medicine
|
| Lab |
Illingworth
|
| Street address |
Centre for Regenerative Medicine
|
| City |
Edinburgh |
| State/province |
Midlothian |
| ZIP/Postal code |
EH16 4UU |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL22129 |
| Series (1) |
| GSE84168 |
Identification of a novel long-range enhancer driving Sonic Hedgehog expression in neural progenitor cells |
|
