|
| Status |
Public on Oct 10, 2012 |
| Title |
Human ESC LaminB1 replicate2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Dam-only DamID DNA from Embryonic Stem Cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: SHEF-2
treatment protocol: infection with lentivirus expressing Dam only
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
1-2x10e5 cells were infected with lenti-virus expressing either Dam-LaminB1 or only Dam. After 48 hours gDNA was isolated with the Qiagen Blood and Tissue kit per the manufacturers protocol. 2.5 μg of gDNA was digested with DpnI (New England Biolabs)), adapters were ligated with T4 ligase ((New England Biolabs)), and the ligation reaction was subsequently digested with DpnII ((New England Biolabs)). 500 ng of the DpnII digested material was used as input for a PCR amplification of methylated fragments (MePCR), see Vogel et al., 2008, Nature Protocols for details. MePCR fragments were purified by Qiaquick Spin columns (Qiagen).
|
| Label |
Cy5
|
| Label protocol |
1.5 µg MePCR DNA was directly labeled by Klenow (New England Biolabs) random priming with Cy3/Cy5 nonamers per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html). Labeling time was extended to 12 hours to increase yield.
|
| |
|
| Channel 2 |
| Source name |
Dam-LaminB1 DamID DNA from Embryonic Stem Cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: SHEF-2
treatment protocol: infection with lentivirus expressing Dam-LaminB1
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
1-2x10e5 cells were infected with lenti-virus expressing either Dam-LaminB1 or only Dam. After 48 hours gDNA was isolated with the Qiagen Blood and Tissue kit per the manufacturers protocol. 2.5 μg of gDNA was digested with DpnI (New England Biolabs)), adapters were ligated with T4 ligase ((New England Biolabs)), and the ligation reaction was subsequently digested with DpnII ((New England Biolabs)). 500 ng of the DpnII digested material was used as input for a PCR amplification of methylated fragments (MePCR), see Vogel et al., 2008, Nature Protocols for details. MePCR fragments were purified by Qiaquick Spin columns (Qiagen).
|
| Label |
Cy3
|
| Label protocol |
1.5 µg MePCR DNA was directly labeled by Klenow (New England Biolabs) random priming with Cy3/Cy5 nonamers per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html). Labeling time was extended to 12 hours to increase yield.
|
| |
|
| |
| Hybridization protocol |
The labeled MePCR DNA was precipitated with 0.1 volume 5M NaCl and 1 volume isopropanol, 80 μg per sample was combined into 33.6 μl water, and mixed with 2.4 μl Alignment oligo, 24 μl hybridization component A, and 60 μl 2x Hybridization Buffer. The entire 120 μl was used for hybridization in Tecan hybridization stations for 16-18 h at 42C. Arrays were then washed in 42 °C Nimblegen washbuffer1, and room temperature Nimblegen washbuffer 2 and 3. All buffers and washes were completed using manufacturer's protocols (http://www.nimblegen.com/products/lit/lit.html)
|
| Scan protocol |
Microarrays were scanned in a Agilent Scanner (Model G2505C, Serial number US22502518), which uses Scan Control software (Version A.8.1.3). Slides were first placed in a slide holder before putting it in the Scanner Carousel, which was installed in the scanner. Slides were double-pass scanned at maximum PMT for each channel, at a 2 um per pixel resolution.
|
| Description |
hESC2
|
| Data processing |
Arrays were quantified with NimbleScan 2.5. Raw .pair files were read with the Ringo package for R and the loess normalized. Subsequently, single channel data was obtained and subjected to quantile-quantile normalisation over all available cell types and replicates.
The score was calculated as the log2 Dam-LaminB1: Dam ratio of the loess-quantile normalized data.
|
| |
|
| Submission date |
Jun 17, 2010 |
| Last update date |
Oct 26, 2012 |
| Contact name |
Bas van Steensel |
| E-mail(s) |
b.v.steensel@nki.nl
|
| Phone |
+ 31 20 512 2040
|
| Fax |
+31 20 669 1383
|
| URL |
http://www.nki.nl/nkidep/vansteensel
|
| Organization name |
Netherlands Cancer Institute
|
| Department |
division of Molecular Biology
|
| Lab |
van Steensel group
|
| Street address |
Plesmanlaan 121
|
| City |
Amsterdam |
| ZIP/Postal code |
1066 CX |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL10559 |
| Series (1) |
| GSE22428 |
Evolutionary conservation of nuclear lamina-genome interactions |
|
