|
| Status |
Public on May 28, 2009 |
| Title |
Bcell hScc1-2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
hScc1ChIP DNA from healthy human Bcell
|
| Organism |
Homo sapiens |
| Characteristics |
Asynchronous culture
|
| Treatment protocol |
Normal condition
|
| Growth protocol |
RPMI+20%FBS
|
| 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. Genomic DNA was obtained by sonication. Chromatin immunoprecipitation was performed using hScc1 antibody. 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 |
End labelled with biotin (biotin-11-ddATP) using Terminal Transferase as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998).
|
| |
|
| Channel 2 |
| Source name |
INPUT DNA from healthy human Bcell
|
| Organism |
Homo sapiens |
| Characteristics |
Asynchronous culture
|
| Treatment protocol |
Normal condition
|
| Growth protocol |
RPMI+20%FBS
|
| 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. Genomic DNA was obtained by sonication. Chromatin immunoprecipitation was performed using hScc1 antibody. 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 |
End labelled with biotin (biotin-11-ddATP) using Terminal Transferase as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998).
|
| |
|
| |
| 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). Washing and scanning protocol provided by Affymetrix was performed automatically on a fluidics station (GeneChip fluidics station 450, Affymetrix).
|
| Scan protocol |
Arrays were scanned using the Genechip Scanner3000 7G following the library array description.
|
| Description |
Cohesin distribution in Bcells
|
| 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 18 (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-6. For ChIP-chip analysis, we use two IP .CEL files and two WCE .CEL files (they are duplicated experiments) to make one profile (see sample.txt file appended below).
|
| |
|
| Submission date |
Aug 28, 2008 |
| Last update date |
May 28, 2009 |
| Contact name |
Katsuhiko Shirahige |
| E-mail(s) |
kshirahi@iam.u-tokyo.ac.jp
|
| 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 |
GPL7223 |
| Series (1) |
| GSE12603 |
Impaired Cohesin Loading in Cornelia de Lange Syndrome Results in a Highly Specific Pattern of Dysregulated Genes |
|
