|
| Status |
Public on Nov 05, 2008 |
| Title |
E.ch-p200 HG18 1 of 2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
p200 ChIP DNA from highly infected (95%) THP-1cells by Ehrlichia chaffeensis
|
| Organism |
Homo sapiens |
| Characteristics |
Age: 7 days infection in culture
Tissue: THP-1
Antibody: anti-p200
|
| Growth protocol |
THP-1 cells were cultured for 7 days in the presence of E. chaffeensis
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP was performed on highly infected (95%) THP-1 cells seven days after infection using ChIP-IT Express kit (Active Motif, Carlsbad, CA) with anti-E. chaffeensis p200 antibody and normal human IgG according to the manufacturer’s instructions. Amplicons were prepared by adapting the standard protocol for whole-genome amplification using the GenomePlex WGA kit (Sigma-Aldrich) except the initial random fragmentation step was eliminated and DNA from entire ChIP and input was amplified.
|
| Label |
Cy5
|
| Label protocol |
1 µg ChIP DNA and input DNA were directly labeled by Klenow (New England Biolabs) random priming with Cy5 nonamers and Cy3 nonamersper based on the manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
|
| |
|
| Channel 2 |
| Source name |
Input DNA from uninfected THP-1 cells
|
| Organism |
Homo sapiens |
| Characteristics |
Antibody: no
Age: 7 days in culture,
|
| Growth protocol |
THP-1 cells were cultured for 7 days
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP was performed on highly infected (95%) THP-1 cells seven days after infection using ChIP-IT Express kit (Active Motif, Carlsbad, CA) with anti-E. chaffeensis p200 antibody and normal human IgG according to the manufacturer’s instructions. Amplicons were prepared by adapting the standard protocol for whole-genome amplification using the GenomePlex WGA kit (Sigma-Aldrich) except the initial random fragmentation step was eliminated and DNA from entire ChIP and input was amplified.
|
| Label |
Cy3
|
| Label protocol |
1 µg ChIP DNA and input DNA were directly labeled by Klenow (New England Biolabs) random priming with Cy5 nonamers and Cy3 nonamersper based on the manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
|
| |
|
| |
| Hybridization protocol |
The labeled ChIP DNA was precipitated with 0.1 volume 5M NaCl and 1 volume isopropanol, and hybridized in 45 ul of buffer containing 20% formamide, 1.2 M betaine, 0.1 ug/ul herring sperm DNA and 10 ug of human COT1 DNA (Invitrogen). Arrays were hybridized in Maui hybridization stations for 16-18 h at 42C, and then washed in 42C 0.2% SDS/0.2x SSC, room temperature 0.2x SSC, and 0.05x SSC. Hybridization buffers and washes were completed using manufacturer's protocols (http://www.nimblegen.com/products/lit/lit.html)
|
| Scan protocol |
Arrays were scanned on an Axon 4000B scanner per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
|
| Description |
Chip-chip p200 infected THP1 cells
|
| Data processing |
A two-array set representing whole-genome promoter (5 kb) tiling, containing 385,000 probes (50- to 75-mer) from ~60,000 human transcripts was obtained from Nimblegen Systems. Labeling and hybridization of E. chaffeensis ChIP samples was performed by Nimblegen Systems. For each spot on the array, log2-ratio of Cy5 labeled p200 IP sample versus the Cy3-labeled input DNA sample (not taken through immunoprecipitation steps) was calculated. The intensity ratio of p200 IP to input DNA was plotted versus genomic position to identify regions where increased signal (i.e. DNA fragment enrichment) was observed relative to the control sample. Positions identified as p200 binding sites on the human 5 kb promoter array were identified by using NimbleScan, and peaks detected by searching for four or more probes above the specified cutoff values, ranging from 90% to 15% using a 500 bp sliding window. The cutoff value is a percentage of a hypothetical maximum (mean ± standard deviation), and the ratio data was randomized 20 times to determine the probability of false positives. Each peak was then assigned a false discovery rate (FDR) score based on the randomization.
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| |
|
| Submission date |
Nov 04, 2008 |
| Last update date |
Nov 04, 2008 |
| Contact name |
Bing Zhu |
| E-mail(s) |
bizhu@utmb.edu
|
| Phone |
409-747-2005
|
| Organization name |
utmb
|
| Street address |
301 University Boulevard
|
| City |
Galveston |
| State/province |
TX |
| ZIP/Postal code |
77550 |
| Country |
USA |
| |
|
| Platform ID |
GPL6325 |
| Series (1) |
| GSE13464 |
ChIP-chip from Ehrlichia chaffeensis-infected THP-1 cells with ankyrin repeat-containing protein, p200 |
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