|
| Status |
Public on Jul 19, 2010 |
| Title |
B6CBA-Fibroblast1 input, methyl-depleted |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Input fraction from B6CBA-Fibroblast1
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Fibroblasts
strain: B6/CBA
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA (gDNA) were isolated using DNeasy kit (Qiagen) according to the manufacturer’s protocol. For each sample, 5 µg of genomic DNA was sheared into 1.6-3kb DNA size fragments, digested with McrBC, and fractionated on a 1% agarose gel alongside the sheared input, methyl-depleted (UT) fraction to enrich for the methyl-depleted (MD) fraction. The UT and MD fractions were purified and whole genome amplified using the WGA2 kit (Sigma) according to the manufacturer's protocol.
|
| Label |
cy3
|
| Label protocol |
2 µg of DNA was directly labeled by Klenow (New England Biolabs) random priming with Cy3 nonamers for the input, methyl-depleted (UT) fraction and Cy5 nonamers for the methyl-depleted (MD) fraction per manufacturer's protocol (http://www.nimblegen.com/products/lit/methylation_userguide_v5p0.pdf).
|
| |
|
| Channel 2 |
| Source name |
Methyl-depleted fraction from B6CBA-Fibroblast1
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Fibroblasts
strain: B6/CBA
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA (gDNA) were isolated using DNeasy kit (Qiagen) according to the manufacturer’s protocol. For each sample, 5 µg of genomic DNA was sheared into 1.6-3kb DNA size fragments, digested with McrBC, and fractionated on a 1% agarose gel alongside the sheared input, methyl-depleted (UT) fraction to enrich for the methyl-depleted (MD) fraction. The UT and MD fractions were purified and whole genome amplified using the WGA2 kit (Sigma) according to the manufacturer's protocol.
|
| Label |
cy5
|
| Label protocol |
2 µg of DNA was directly labeled by Klenow (New England Biolabs) random priming with Cy3 nonamers for the input, methyl-depleted (UT) fraction and Cy5 nonamers for the methyl-depleted (MD) fraction per manufacturer's protocol (http://www.nimblegen.com/products/lit/methylation_userguide_v5p0.pdf).
|
| |
|
| |
| Hybridization protocol |
The labeled DNA was precipitated with 0.1 volume 5M NaCl and 1 volume isopropanol, and hybridized in NimbleGen Hybridization solution master mix. Arrays were hybridized in Maui hybridization stations for 16-20 h at 42C, and washes were completed using manufacturer's protocols (http://www.nimblegen.com/products/lit/methylation_userguide_v5p0.pdf).
|
| Scan protocol |
Arrays were scanned on a GenePix 4000B scanner per manufacturer's protocol (http://www.nimblegen.com/products/lit/methylation_userguide_v5p0.pdf).
|
| Description |
100512_MM8_CHARM_HX1.txt
|
| Data processing |
Arrays were processed using Nimblegen's standard protocol for Nimblescan 2.4 software (http://www.nimblegen.com/products/lit/methylation_userguide_v5p0.pdf).
|
| |
|
| Submission date |
Jul 14, 2010 |
| Last update date |
Jul 16, 2010 |
| Contact name |
Akiko Doi |
| E-mail(s) |
adoi@jhmi.edu
|
| Phone |
410-614-3478
|
| Organization name |
Johns Hopkins University School of Medicine
|
| Department |
Medicine
|
| Street address |
855 N Wolfe St, Rangos 580
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21205 |
| Country |
USA |
| |
|
| Platform ID |
GPL10683 |
| Series (1) |
| GSE22851 |
DNA methylation data from mouse iPS cells, ES cells, and ntES cells |
|
