|
| Status |
Public on Jan 31, 2016 |
| Title |
lung Replicate 2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
lung_2_INPUT
|
| Organism |
Mus musculus |
| Characteristics |
tissue: lung
gender: Female
age: 3 months
sample type: input
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
mouse (40µg) DNA was sonicated to an average fragment size of 300–1000 bp, precipitated with 400 mM NaCl, 2 volumes of ethanol and 1 µl glycogen. 1.5 µg were set aside as the Input fraction. DNA was denatured and anti-5-methylcytidine monoclonal antibody (10 µl for 5 µg) was added and incubated on a rotator at 4°C overnight. 40 µl Dynabeads (Sheep anti-Mouse IgG) were prewashed with 0.1% BSA/PBS and added to the DNA. The DNA was then washed 3 times and Ab-bound DNA resuspended and extracted with proteinase K, phenol-chloroform and ethanol precipitation.
|
| Label |
Cy3
|
| Label protocol |
The Input and Bound DNAs were labeled using the Agilent Technologies Genomic Labeling kit Plus according to the manufacturer’s instructions. Briefly, 300ng of each sample was labeled with Cyanine-3 or Cyanine-5 dUTP and purified using microcon YM 30 columns.
|
| |
|
| Channel 2 |
| Source name |
lung_2_BOUND
|
| Organism |
Mus musculus |
| Characteristics |
tissue: lung
gender: Female
age: 3 months
sample type: bound
antibody: anti-5-methylcytidine monoclonal antibody
antibody manufacturer: Diagenode
antibody catalog #: MAb-081-100
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
mouse (40µg) DNA was sonicated to an average fragment size of 300–1000 bp, precipitated with 400 mM NaCl, 2 volumes of ethanol and 1 µl glycogen. 1.5 µg were set aside as the Input fraction. DNA was denatured and anti-5-methylcytidine monoclonal antibody (10 µl for 5 µg) was added and incubated on a rotator at 4°C overnight. 40 µl Dynabeads (Sheep anti-Mouse IgG) were prewashed with 0.1% BSA/PBS and added to the DNA. The DNA was then washed 3 times and Ab-bound DNA resuspended and extracted with proteinase K, phenol-chloroform and ethanol precipitation.
|
| Label |
Cy5
|
| Label protocol |
The Input and Bound DNAs were labeled using the Agilent Technologies Genomic Labeling kit Plus according to the manufacturer’s instructions. Briefly, 300ng of each sample was labeled with Cyanine-3 or Cyanine-5 dUTP and purified using microcon YM 30 columns.
|
| |
|
| |
| Hybridization protocol |
The labeled Input and Bound DNAs were combined and hybridized for 60 h at 67°C in 15% deionized formamide to a CpG island microarray using the Agilent aCGH hybridization buffer and protocol.
|
| Scan protocol |
Arrays were washed according to the Agilent aCGH protocol, immediately scanned on an Agilent microarray scanner and processed using Agilent Feature extraction software version 9.5.
|
| Data processing |
Probe log ratio Cy5/Cy3 signals were Loess normalized and transformed into Z-scores according to their Tm.
|
| |
|
| Submission date |
Sep 02, 2010 |
| Last update date |
Jan 31, 2016 |
| Contact name |
Deborah Nejman |
| Organization name |
Hebrew University
|
| Street address |
Ein Kerem
|
| City |
Jerusalem |
| ZIP/Postal code |
91120 |
| Country |
Israel |
| |
|
| Platform ID |
GPL7099 |
| Series (1) |
| GSE23953 |
Detection of aberrant DNA methylation in undifferentiated and differentiated mouse ES cells compared to normal tissues |
|
