|
| Status |
Public on Feb 15, 2014 |
| Title |
HumanHT1080 BAF_repl1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Dam-only DamID DNA from HT1080
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HumanHT1080
expression: Dam
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For the Dam-BAF 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 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). For the Dam-Lamin2 sample: Same as for the Dam-BAF sample except that Dam-Lamin B2 was introduced by a Lipofectamine (Invitrogen) transfection of 4μg plasmid.
|
| 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.
|
| |
|
| Channel 2 |
| Source name |
Dam-BAF DamID DNA from HT1080
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HumanHT1080
expression: Dam-BAF
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For the Dam-BAF 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 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). For the Dam-Lamin2 sample: Same as for the Dam-BAF sample except that Dam-Lamin B2 was introduced by a Lipofectamine (Invitrogen) transfection of 4μg plasmid.
|
| 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.
|
| |
|
| |
| 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 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 |
ch1 raw data file= CMF_355053_S01_Cy3.pair
ch2 raw data file= CMF_355053_S01_Cy5.pair
|
| Data processing |
Arrays were quantified with NimbleScan 2.5. Raw .pair files were read with the Ringo package for R and the loess normalized. The score was calculated as the log2 Dam-LaminB2 or Dam-BAF: Dam ratio of the normalized data.
|
| |
|
| Submission date |
Feb 14, 2014 |
| Last update date |
Feb 15, 2014 |
| 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) |
| GSE55066 |
Stochastic genome-nuclear lamina interactions: Modulating roles of Lamin A and BAF |
|
