|
| Status |
Public on Jan 30, 2008 |
| Title |
hSmc3 distribution in M-phase (ENCODE) |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Chromatin Immuno-precipitated DNA
|
| Organism |
Homo sapiens |
| Characteristics |
HeLa cells. Anti-hSmc3 antibody was used for Chromatin immuno-precipitation.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells at 70-80% confluency were crosslinked with 1% formaldehyde for 10 min and after quenching with 125 mM glycine prepared for ChIP. Chromatin immunoprecipitation was performed using Scc1, Smc3, CTCF, SA2 and control antibodies. The lysate was incubated with the antibodies over night. Then the pre-absorbed protein A Affiprep beads (Bio-Rad) were added and incubated for 2 hours at 4 degree. The beads were washed several times and eluted for 20 min at 65 degree with elution buffer (50 mM Tris, 10 mM EDTA, 1 % SDS). The eluate from the beads was treated with Proteinase K and decrosslinked at 65°C over night. Contaminating RNA was removed by RNAse treatment. Then the sample was further purified by phenol-chloroform extraction and one additional purification step using the PCR purification kit (Qiagen).
|
| Label |
Biotin-11-ddATP
|
| Label protocol |
DNA was amplified by IVT (In vitro transcription) method. For the labeling, DNA was fragmented by DNaseI. DNA was end-labeled by Terminal Transferase with biotin‐N11‐ddATP (NEL508).
|
| |
|
| Channel 2 |
| Source name |
Whole cell extract (WCE) fraction used for the normalization of ChIP fraction.
|
| Organism |
Homo sapiens |
| Characteristics |
HeLa cells
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
same as channel 1
|
| Label |
Biotin-11-ddATP
|
| Label protocol |
same as channel 1
|
| |
|
| |
| Hybridization protocol |
Each sample was hybridized to the array in 200 ul containing 1XHybridization buffer (Affymetrix), 50pM Control oligonucleotide (oligo B2, Affymetrix), Herring Sperm DNA (0.1mg/ml), Acetylated BSA (0.5mg/ml), and 7% DMSO. Samples were denatured at 100C for 10 minutes, and then put on ice before being hybridized for 16 hours at 45C in an hybridization oven (GeneChip Hybridization Oven 640, Affymetrix).
|
| Scan protocol |
Arrays were scanned using the Genechip Scanner3000 7G following the library array description.
|
| Description |
hSmc3 distribution in M-phase. Wce fraction used for the analyses of SA2 in G2 phase was used for normalization.
|
| Data processing |
Array intensity data from duplicated experiments of ChIP and WCE fraction were compared by MAT (Model-based Analysis of Tiling-array) algorithm, using the software provided by the laboratory of X.Shirley Liu (http://chip.dfci.harvard.edu/~wli/MAT/). We calculated MAT score and mapped the results to genomic positions in human genome assembly hg 17 (NCBI Build 36). Bandwidth, MaxGap, and MinProbe parameters were set to 250, 1000, and 12, respectively. Cutoff threshold p-values were set to 1.0e-10, 1.0e-8, and 5.0e-8 for ENCODE1.0, 2.0, and Human Tiling 1.0R, respectively.
For ChIP-chip analysis, we use two IP .CEL files and two WCE .CEL files (they are duplicated experiments) to make one profile.
|
| |
|
| Submission date |
Nov 16, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Katsuhiko Shirahige |
| E-mail(s) |
[email protected]
|
| Phone |
+81-3-5842-0756
|
| Fax |
+81-3-5842-0757
|
| URL |
http://www.iam.u-tokyo.ac.jp/chromosomeinformatics/
|
| Organization name |
The University of Tokyo
|
| Department |
Research Center for Epigenetic Disease
|
| Lab |
Laboratory of Genome Structure and Function
|
| Street address |
1-1-1 Yayoi
|
| City |
Bunkyo-ku |
| State/province |
Tokyo |
| ZIP/Postal code |
113-0032 |
| Country |
Japan |
| |
|
| Platform ID |
GPL6130 |
| Series (1) |
| GSE9613 |
Cohesin mediates transcriptional insulation by CCCTC-binding factor |
|
