|
| Status |
Public on Aug 20, 2010 |
| Title |
H929 2X |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
MIRA-enriched DNA
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: H929 methylated DNA fraction
cycles: 2X WGA
cell line: H929
|
| Growth protocol |
DMEM + 10% FBS
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was purified from Raji and H929 were sonicated using a Bioruptor (Diagenode) to generate 200-500 bp fragments. Methylated CpG DNA fragments were enriched from 200 ng of sonicated DNA using the MethylCollector Ultra Kit (Actif Motif) following the manufacturer’s protocol.
|
| Label |
cy5
|
| Label protocol |
Input and methylated CpG DNA fragments were amplified (WGA2, Sigma), labeled with Cy3 and Cy5 random nonomers (Trilink Biotechnologies) following NimbleGen’s protocol.
|
| |
|
| Channel 2 |
| Source name |
Input DNA
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: H929 input DNA
cycles: 2X WGA
cell line: H929
|
| Growth protocol |
DMEM + 10% FBS
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was purified from Raji and H929 were sonicated using a Bioruptor (Diagenode) to generate 200-500 bp fragments. Methylated CpG DNA fragments were enriched from 200 ng of sonicated DNA using the MethylCollector Ultra Kit (Actif Motif) following the manufacturer’s protocol.
|
| Label |
cy3
|
| Label protocol |
Input and methylated CpG DNA fragments were amplified (WGA2, Sigma), labeled with Cy3 and Cy5 random nonomers (Trilink Biotechnologies) following NimbleGen’s protocol.
|
| |
|
| |
| Hybridization protocol |
Samples were hybridized onto a NimbleGen 2.1M Deluxe Human Promoter Array following NimbleGen’s protocol.
|
| Scan protocol |
The microarray slides were scanned using the Agilent G2565BA DNA Microarray Scanner. Images were processed using the NimbleScan software.
|
| Description |
MIRA-chip from H929
|
| Data processing |
The log2(Cy5) values were normalized using loess curve fitting conditioning on GC content as described in BMC Bioinformatics 2009, 10:173 doi:10.1186/1471-2105-10-173. These signal values were further quantile normalized to adjust for between sample variations. This method accounts for CG effect and also incorporates cy3 data as following. First we bin all probes according to their GC content defined as a ratio of number of C and G nucleotides to the total number of nucleotides in the probe sequence. We used overall variability in GC content values to compute bin width according to zero-stage rule described by Wand (1997). The bins with fewer probes are then merged to make sure that each bin contains at least 500 probes. Within each bin we use loess regression to predict log2(cy5) values as a smooth function of log2(cy3) values. The residuals (difference between observed and predicted value) from this loess fit are then divided by their mean absolute deviations to compute normalized signal.
|
| |
|
| Submission date |
Jul 12, 2010 |
| Last update date |
Jul 13, 2010 |
| Contact name |
Deepak Mav |
| Organization name |
Sciome LLC
|
| Street address |
2 Davis Drive
|
| City |
Research Triangle Park |
| State/province |
NC |
| ZIP/Postal code |
27709 |
| Country |
USA |
| |
|
| Platform ID |
GPL10671 |
| Series (1) |
| GSE22884 |
DNA methylation prevents CTCF-mediated silencing of the oncogene BCL6 in B cell lymphomas |
|
