|
| Status |
Public on Aug 24, 2012 |
| Title |
CELL LINE g2mr5 GluMspI |
| Sample type |
genomic |
| |
|
| Source name |
Hypothalamic neurons (mHypoA-2/24 cell line)
|
| Organism |
Mus musculus |
| Characteristics |
age (y): NA
Sex: NA
post-mortem interval: NA
tissue: Hypothalamic neurons (mHypoA-2/24 cell line)
cell line: mHypoA-2/24
cell cycle stage: G2
enzymatic digestion: 200 ng of glucosylated 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.
|
| 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
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| |
|
| 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 |
DNA was extracted from G2 cell cycle stage cells of murine neuronal cell line (mHypoA-2/24).
|
| 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.
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| |
|
| Submission date |
Aug 16, 2012 |
| Last update date |
Aug 24, 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 |
GPL6445 |
| 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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