|
| Status |
Public on Aug 24, 2012 |
| Title |
SAHA 15 30 TYPE4 MspI |
| Sample type |
genomic |
| |
|
| Source name |
Transformed B-lymphocytes (GM10851 cell line)
|
| Organism |
Homo sapiens |
| Characteristics |
age (y): NA
Sex: NA
post-mortem interval: NA
tissue: Transformed B-lymphocytes (GM10851 cell line)
cell line: GM10851
enzymatic digestion: 200 ng of native sheared DNA were subjected to 10 U of MspI digestion in a final volume of 20 µl for 12 h at 37ºC (50 fold surplus of the enzyme over the DNA substrate), followed by enzyme inactivation at 80ºC for 10 min.
|
| Biomaterial provider |
Coriell; http://ccr.coriell.org/Sections/Search/Search.aspx?PgId=165&q=GM10851
|
| Treatment protocol |
No treatment for human and mouse tissues. Only one human cell line underwent SAHA treatment protocol; stated in the sample description.
|
| Growth protocol |
Human and mouse untreated tissues were analyzed in this study
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted applying standard phenol chloroform and isopropanol precipitation method. Genomic DNA was then sheared to 200bp fragment length on Covaris S2 sonifier (Covaris, USA); blunt ended and ligated to adaptors before subjecting for glucosylation and restriction enzyme digestion. Glucosylation reaction was performed in presence of 200 µM uridine-5’-diphospho-α -D-glucose (UDP-Glc, Sigma) and 80ng BGT (Lariviere, Kurzeck et al. 2002) in 100mM Tris-HCl (pH 8.0) and 25mM MgCl2 for 3 h at 37ºC.
|
| Label |
biotin
|
| Label protocol |
9 ug of PCR amplified DNA products were enzymatically fragmented and labeled using the WT Labeling Kit (Affymetrix) to add biotinylated compound to the ends of the fragments
|
| |
|
| Hybridization protocol |
Approximately 9 μg of DNA was hybridzed per array using the Affymetrix hybridization kit. All array sets were hybridized for 16 hours at 45° at 60rpm using an Affymetrix hybridization oven.
|
| Scan protocol |
Arrays were scanned on an Affymetrix Gene Chip Scanner 3000 7G
|
| Description |
Transformed human B-lymphocyte cells (GM10851, Coriell Cell Repositories) were treated with the histone deacteylase inhibitor suberoylanilide hydroxamic acid. Treated and untreated DNA was then separately processed using the standard protocol of this study (3 conditions, including genomic DNA glucosylation followed by restriction enzyme digestion).
|
| Data processing |
We distinguished between three types of probes on the tiling microarray: “target probes”, which contain the restriction site CCGG, “flanking probes”, which do not contain the recognition sequence but could lie up to 200 bp upstream or downstream of a target probe (sheared DNA fragments were of average 200 bp), and “non-target probes” that neither contain a target sequence nor are within 200 bp (on either side) of the target sequence. After comparing different array preprocessing algorithms, we chose to use a sequence-based algorithm after Potter et al (Supplementary Note). The model (Supplementary Note, equation 1) was applied to non-target probes matched proportionally to target probes by GC content. Normalized intensities were obtained by subtracting the fitted value from raw probe intensities, resulting in a normal distribution of probe-level intensities. Each chip was independently normalized. All downstream analyses were carried out at the single-probe level (i.e. without windowing or peak-calling) and solely on target probes. Probes overlapping repeats were excluded. Probes were required to have a ‘CCGG’ in both the Affymetrix probe sequence and the chromosomal sequence downloaded from UCSC; probes that did not meet this criterion were excluded.
EXP2_Human_BrainLiver_sample_metadata.xls, HumanBrain_sample_metadata.xls
BED files of normalized intensities for target probes. Unless otherwise specified, probes overlapping repeats are excluded. Unless otherwise specified, target probes that did not contain a CCGG in the UCSC genome sequence were also excluded.
|
| |
|
| Submission date |
Aug 16, 2012 |
| Last update date |
Oct 17, 2012 |
| Contact name |
Shraddha Pai |
| E-mail(s) |
shraddha.pai@utoronto.ca
|
| Organization name |
University of Toronto
|
| Street address |
160 College Street, Room 602
|
| City |
Toronto |
| State/province |
ON |
| ZIP/Postal code |
M5S 3E1 |
| Country |
Canada |
| |
|
| Platform ID |
GPL4916 |
| Series (2) |
| GSE40166 |
5-hmC in the brain: abundance in synaptic genes and differences at the exon-intron boundary (tiling array) |
| GSE40167 |
5-hmC in the brain: abundance in synaptic genes and differences at the exon-intron boundary |
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