|
| Status |
Public on Apr 25, 2018 |
| Title |
Human K562 LaminB1 replicate2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Dam-LaminB1 DamID DNA from K562 Cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: K-562 (ATCC CCL-243)
expression: Dam-LaminB1
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
1.5x10e6 cells were infected with lenti-virus expressing either Dam-LaminB1 or only Dam. After 48 hours gDNA was isolated with the Bioline ISOLATE II Genomic DNA Kit per the manufacturers protocol. The DNA was precipitated with ethanol. 1.5µg genomic DNA was treated for 1 hour with 5 units of Antarctic phosphatase (New England Biolabs, cat nr M0289S) in 20µl of 1X Antarctic Phosphatase Reaction Buffer (New England Biolabs) followed by ethanol precipitation. 100-500 ng 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)). 50-200 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 ISOLATE II PCR and Gel Kit (Bioline).
|
| Label |
Cy5
|
| Label protocol |
2.0 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 2 hours to increase yield.
|
| |
|
| Channel 2 |
| Source name |
Dam-only DamID DNA from K562 Cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: K-562 (ATCC CCL-243)
expression: Dam
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
1.5x10e6 cells were infected with lenti-virus expressing either Dam-LaminB1 or only Dam. After 48 hours gDNA was isolated with the Bioline ISOLATE II Genomic DNA Kit per the manufacturers protocol. The DNA was precipitated with ethanol. 1.5µg genomic DNA was treated for 1 hour with 5 units of Antarctic phosphatase (New England Biolabs, cat nr M0289S) in 20µl of 1X Antarctic Phosphatase Reaction Buffer (New England Biolabs) followed by ethanol precipitation. 100-500 ng 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)). 50-200 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 ISOLATE II PCR and Gel Kit (Bioline).
|
| Label |
Cy3
|
| Label protocol |
2.0 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 2 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
|
| 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 |
Feb 18, 2015 |
| Last update date |
Apr 25, 2018 |
| Contact name |
Andrew S Belmont |
| E-mail(s) |
asbel@illinois.edu
|
| Phone |
(217) 244-2311
|
| Organization name |
University of Illinois at Urbana-Champaign
|
| Street address |
601 S Goodwin Ave, CLSL B509
|
| City |
Urbana |
| State/province |
IL |
| ZIP/Postal code |
61801 |
| Country |
USA |
| |
|
| Platform ID |
GPL10559 |
| Series (2) |
| GSE66018 |
TSA-Seq Mapping of Nuclear Genome Organization [array] |
| GSE66019 |
TSA-Seq Mapping of Nuclear Genome Organization |
|
