|
| Status |
Public on May 05, 2011 |
| Title |
KM-H2_Acet_ChIP |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
KM-H2 H3 K9/14 ChIP-DNA
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: KM-H2
cell type: Hodgkin lymphoma cells
antibody: anti-acetyl H3K9/14 rabbit polyclonal
antibody manufacturer: Upstate Chemicon
antibody catalog #: 06-599
|
| Growth protocol |
Three Hodgkin lymphoma cell lines (L1236, KM-H2, L428), three plasma cell myeloma cell lines (L363, U266, LP-1), and three B-cell lines (SU-DHL4, SU-DHL6, Namalwa) were cultured with 5% CO2 in RPMI 1640 (PAA, Pasching, Austria) supplemented with 10% fetal bovine serum (PAA) at 37°C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The cell lines were used for ChIP following the protocol developed by the group of Young (PMID 17406303) with minor modifications. For each ChIP experiment, 10 µg of anti-acetyl-histone H3 (Lys9 + Lys14) antibody (06-599; Upstate Chemicon, Temecula, CA, USA) was employed. All experiments were performed in duplicates or triplicates. A detailed protocol is provided as supplementary information (Supplementary protocol S1) in our publication related to this GEO data.
|
| Label |
biotin
|
| Label protocol |
Approximately 250 ng of ChIP-DNA was used as a template for ligation-mediated linear amplification according to the protocol of Young et al. to obtain a sufficient amount of labeled DNA for chip hybridization. The GeneChIP WT double-stranded DNA terminal labeling kit (Affymetrix) was used for DNA-fragmentation according to the manufacturer’s instructions.
|
| |
|
| Channel 2 |
| Source name |
KM-H2 Input DNA
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: KM-H2
cell type: Hodgkin lymphoma cells
antibody: none
|
| Growth protocol |
Three Hodgkin lymphoma cell lines (L1236, KM-H2, L428), three plasma cell myeloma cell lines (L363, U266, LP-1), and three B-cell lines (SU-DHL4, SU-DHL6, Namalwa) were cultured with 5% CO2 in RPMI 1640 (PAA, Pasching, Austria) supplemented with 10% fetal bovine serum (PAA) at 37°C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The cell lines were used for ChIP following the protocol developed by the group of Young (PMID 17406303) with minor modifications. For each ChIP experiment, 10 µg of anti-acetyl-histone H3 (Lys9 + Lys14) antibody (06-599; Upstate Chemicon, Temecula, CA, USA) was employed. All experiments were performed in duplicates or triplicates. A detailed protocol is provided as supplementary information (Supplementary protocol S1) in our publication related to this GEO data.
|
| Label |
biotin
|
| Label protocol |
Approximately 250 ng of ChIP-DNA was used as a template for ligation-mediated linear amplification according to the protocol of Young et al. to obtain a sufficient amount of labeled DNA for chip hybridization. The GeneChIP WT double-stranded DNA terminal labeling kit (Affymetrix) was used for DNA-fragmentation according to the manufacturer’s instructions.
|
| |
|
| |
| Hybridization protocol |
Reaction mixtures were hybridized to the GeneChip® Human Promoter 1.0R Arrays for 16 h at 45 °C at 60 rpm and stained with streptavidin/phycoerythrin, followed by a biotin-conjugated anti-streptavidin antibody and a second streptavidin/phycoerythrin staining. All liquid handling was carried out by a GeneChip Fluidics Station 450.
|
| Scan protocol |
GeneChips were scanned with the Affymetrix GeneChip Scanner 3000 7G (Affymetrix) and CEL files were generated with the GCOS 1.3 software (Affymetrix).
|
| Description |
Acet H3 K9/14 ChIP, Biological Rep 1-3, KM-H2
|
| Data processing |
Resulting CEL files were analyzed using the Model-based Analysis of Tiling arrays (MAT) algorithm. We used default parameters except for the MaxGap parameter, which was reduced to 50 bp to find acetylated regions at higher resolution. Instead of working with the original Affymetrix BPMAP files, we used the microarray probe mapping to NCBI build 36 provided by the MAT homepage (http://liulab.dfci.harvard.edu/MAT/). This analysis resulted in a list of significantly acetylated regions for each of the nine cell lines.
Bar and Bed files were generated with the Model-based Analysis of Tiling arrays (MAT) software.
|
| |
|
| Submission date |
Apr 07, 2010 |
| Last update date |
May 05, 2011 |
| Contact name |
Volkhard Seitz |
| E-mail(s) |
[email protected]
|
| Organization name |
Charité Universitätsmedizin
|
| Department |
Institute of Pathology
|
| Street address |
Hindenburgdamm 30
|
| City |
Berlin |
| ZIP/Postal code |
12200 |
| Country |
Germany |
| |
|
| Platform ID |
GPL5082 |
| Series (2) |
| GSE21253 |
Classical Hodgkin lymphoma shows epigenetic features of an abortive plasma cellular differentiation: acetyl H3 K9/14 ChIP-chip |
| GSE21254 |
Classical Hodgkin lymphoma shows epigenetic features of an abortive plasma cellular differentiation |
|
