|
| Status |
Public on Sep 29, 2007 |
| Title |
Brain Normal_TspR1-ExoIII-rep1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Brain-MOCK
|
| Organism |
Homo sapiens |
| Characteristics |
This genomic DNA was purchased from BioChain. (Catalog No.: D1234035).
Human, Normal Brain, Male, 41 years old
Genomic DNA was NOT digested with HpaII
|
| Treatment protocol |
Genomic DNA was first digested with TspR I, which created DNA fragments with 9-base 3’ extension and were resistant for exonuclease III digestion. The DNA was NOT digested with methylation-sensitive restriction enzyme Hpa II. Then treatment with exonuclease III and RecJf exonuclease, DNA were purified and labeled with Cy3 and Cy5 fluorescence dyes respectively. The Cy3 hybridization intensity was normalized to Cy5 for comparison among samples.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
This genomic DNA was purchased from BioChain (Catalog No.: D1234035)
|
| Label |
Cy3
|
| Label protocol |
According to the manual of Agilent Oligo Microarray Kit.
|
| |
|
| Channel 2 |
| Source name |
Brain-HpaII
|
| Organism |
Homo sapiens |
| Characteristics |
This genomic DNA was purchased from BioChain. (Catalog No.: D1234035).
Human, Normal Brain, Male, 41 years old
Genomic DNA was NOT digested with HpaII
|
| Treatment protocol |
Genomic DNA was first digested with TspR I, which created DNA fragments with 9-base 3’ extension and were resistant for exonuclease III digestion. The DNA were then digested with methylation-sensitive restriction enzyme Hpa II. After treatment with exonuclease III and RecJf exonuclease, DNA were purified and labeled with Cy3 and Cy5 fluorescence dyes respectively. The Cy3 hybridization intensity was normalized to Cy5 for comparison among samples.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
This genomic DNA was purchased from BioChain (Catalog No.: D1234035)
|
| Label |
Cy5
|
| Label protocol |
According to the manual of Agilent Oligo Microarray Kit.
|
| |
|
| |
| Hybridization protocol |
According to the manual of Agilent protocol (http://www.agilent.com)
|
| Scan protocol |
Scanning was performed as the manual of Agilent protocol (http://www.agilent.com)
|
| Description |
Protected from Exonuclease III digestion by TspRI ends.
1~2μg DNA was digested with methylation sensitive restriction enzyme in 10μl of total volume for 2hrs, 1μl(10units) TspR I was then added and reacted at 65℃ for two hours using hot top PCR machine. 10unit of Exonuclease III enzyme was then added and total volume was brought to 20μl by addition of water and 10X buffer. Reaction was carried out at 30℃ for 1hr. Exonuclease III was heat inactive by 70℃ for 20min. 30units RecJf was added to remove single-strand DNA and the enzyme was inactivated by heating at 65℃ for 20min. DNA was then phenol/chloroform extracted and ethanol precipitate.
|
| Data processing |
The Cy3 hybridization intensity was normalized to Cy5 for comparison among samples. The log2 ratio(log2 Cy5/Cy3) were calculated and compared. Agilent software was used.
|
| |
|
| Submission date |
Aug 31, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Ming-Ta Hsu |
| E-mail(s) |
d88307@ym.edu.tw
|
| Phone |
886-2-28267230
|
| Organization name |
National Yang-Ming University
|
| Street address |
155, Li-Nong St., Sec.2, Peitou, Taipei, Taiwan, R.O.C.
|
| City |
Taipei |
| ZIP/Postal code |
112 |
| Country |
Taiwan |
| |
|
| Platform ID |
GPL4091 |
| Series (1) |
| GSE9015 |
Genome-Wide Mapping of Hypomethylated Sites in Human Genomes |
|
