|
| Status |
Public on Nov 18, 2014 |
| Title |
CON.HET.3.MEDIP |
| Sample type |
genomic |
| |
|
| Source name |
murine hippocampal MeDIP DNA from unstressed 5Htt +/- animals
|
| Organism |
Mus musculus |
| Characteristics |
backgroud strain: C57BL6
treatment: control group
|
| Growth protocol |
The wt and 5-Htt +/- deficient mice ([B6.129(Cg)-Slc6a4tm1Kpl/J]) were housed individually within a temperature-controlled environment (2161uC) with 12 h light/12 h dark cycle (lights on from 7.00 h) and standard rodent chow and water available ad libitum.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted from the tissue using phenol/chloroform/isoamyl alcohol extraction. 300 µl 0.5%-SDS extraction buffer were added to the frozen tissue. The tissue was subsequently homogenized for 30 to 60 sec at 25 Hz at 4°C in the Tissue Lyser (Qiagen, Hilden, Germany) using a metallic bead. After adding 200 µl 0.5%-SDS extraction buffer and 50 µl 10- or 20mg/µl-proteinase K, samples were incubated at 55°C over night/3 h. Following this, samples were incubated with 50 µl 10mg/µl-RNAse A with for 1 h at 37°C and then mixed with 700 µl of phenol/chloroform/isoamyl alcohol mixture (25:24:1). Phases were separated using MaXtract high density tubes (Qiagen) by centrifuging the samples at 14 000 rpm for 5 min at RT. The aqueous phase was then mixed with 700 µl phenol isoamyl alcohol (24:1) and phases were again separated by centrifuging the samples in MaXtract high density tubes as described above. DNA was precipitated with 50 µl sodium acetate and 1000 µl cold absolute ethanol (incubation 10 min at -20°C, centrifugation 20 min at 4°C, 14 000 rpm). The resulting pellet was washed with 500 µl cold 80%-ethanol using the same conditions as for the precipitation, dried at RT for 5 to 30 min and resuspended in 50 µl 1xTE. DNAs of single animals were then pooled, creating 2-3 pools per group. DNA was then sheared using the BiorupterTM UCD-200 (Diagenode, Liège, Belgium) yielding fragments of 300bp size +/- 200bp.Conditions were 20 KHz, 30 sec ON alternated by 30 sec OFF, for 3 x 5 min at low power
|
| Label |
biotin
|
| Label protocol |
Samples (approximately 7 µg of amplified DNA each) were fragmented and terminally labeled with biotin using the GeneChip Mapping 10K Xba kit (Affymetrix).
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| |
|
| Hybridization protocol |
Approximately 7µg of amplified and terminally labeled DNA was hybridized to tiling arrays at 45°C for 16h at 60 rpm. Washing of arrays and staining of bound DNA was achieved with the GeneChipHWS kit in a Fluidics Station 450 (both Affymetrix) using fluidics script FS450_0001.
|
| Scan protocol |
Microarrays were scanned using a GeneChip Scanner 3000 7G (Affymetrix).
|
| Description |
MeDIP unstressed 5Htt +/- mice group 3
|
| Data processing |
Data from MeDIP and input pairs were loess normalized; log2(MeDIP/input) signal log ratios (SLR) were reported in bedGraph files. Coordinates of genomic probes were mapped to the M. musculus MMv37 sequence assembly. Analyses were performed in R (http://www.r-project.org) with the Bioconductor package Starr (http://www.bioconductor.org).
Raw data from CEL files of MeDIP and input pairs of the same sample were loess normalized, followed by calculation of the MeDIP-input signal log ratio (SLR). SLRs between samples were quantiles normalized, followed by a 300bp sliding-window median smoothing along the genomically ordered probe signals. The preprocessed array signals are reported in the "_smoothed.bedGraph" files. The CMARRT algorithm was employed to define MeDIP-enriched autosomal regions in smoothed SLRs of each sample; the ".cmarrt.bed" files contain genomic coordinates of MeDIP-enriched regions. Analyses were performed in R (http://www.r-project.org) with the Bioconductor package Starr (http://www.bioconductor.org).
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| |
|
| Submission date |
Oct 24, 2013 |
| Last update date |
Nov 20, 2014 |
| Contact name |
Claus Juergen Scholz |
| Organization name |
University Hospital Würzburg
|
| Department |
Core Unit SysMed
|
| Street address |
Josef-Schneider-Str. 2
|
| City |
Würzburg |
| ZIP/Postal code |
97080 |
| Country |
Germany |
| |
|
| Platform ID |
GPL5811 |
| Series (1) |
| GSE51634 |
Prenatal stress-induced programming of genome-wide promoter DNA methylation in 5-Htt deficient mice |
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