|
| Status |
Public on Jan 31, 2013 |
| Title |
HumanHT1080 clone11 LaminB1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Dam-only DamID DNA from HT1080
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HT1080
expression: Dam
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For the Dam-only sample: 1-2x10e5 cells were infected with lenti-virus expressing Dam. After 48 hours, gDNA was isolated with the Qiagen Blood and Tissue kit per the manufacturer's 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).
For the Dam-LaminB1 sample: Same as for the Dam-only sample except that Dam-LMNB1 was induced in a stable cell line (Clone11) with 500nM Shield1 for 24 hours.
|
| Label |
Cy3
|
| Label protocol |
1.5 µg MePCR DNA was directly labeled by Klenow (New England Biolabs) random priming with Cy3/Cy5 nonamers per the 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 HT1080
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HT1080 (clone 11)
expression: Dam-LaminB1
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For the Dam-only sample: 1-2x10e5 cells were infected with lenti-virus expressing Dam. After 48 hours, gDNA was isolated with the Qiagen Blood and Tissue kit per the manufacturer's 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).
For the Dam-LaminB1 sample: Same as for the Dam-only sample except that Dam-LMNB1 was induced in a stable cell line (Clone11) with 500nM Shield1 for 24 hours.
|
| Label |
Cy5
|
| Label protocol |
1.5 µg MePCR DNA was directly labeled by Klenow (New England Biolabs) random priming with Cy3/Cy5 nonamers per the 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. 50 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 42°C. Arrays were then washed in 42°C NimbleGen washbuffer1, and room temperature NimbleGen washbuffer 2 and 3. All buffers and washes were completed using the manufacturer's protocols (http://www.nimblegen.com/products/lit/lit.html).
|
| Scan protocol |
Microarrays were scanned in an 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 |
Sample 3
|
| Data processing |
Arrays were quantified with NimbleScan 2.5. Raw .pair files were read with the Ringo package for R and then loess normalized. The score was calculated as the log2 Dam-LaminB1: Dam ratio of the normalized data.
|
| |
|
| Submission date |
Aug 13, 2012 |
| Last update date |
Jan 31, 2013 |
| 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 (2) |
| GSE40094 |
Stochastic genome - nuclear lamina contacts are linked to histone H3K9 dimethylation (methylation data) |
| GSE40112 |
Stochastic genome - nuclear lamina contacts are linked to histone H3K9 dimethylation |
|
