|
| Status |
Public on Mar 10, 2018 |
| Title |
Mesial Temporal Lobe Epilepsy Tissue 3 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Mesial Temporal Lobe Epilepsy tissue_MeDIP-WGA
|
| Organism |
Homo sapiens |
| Characteristics |
subject status: Mesial temporal epilepsy (MTLE) patient
age: 14
gender: MALE
tissue: brain
|
| Biomaterial provider |
DR. P. SARAT CHANDRA DEPT OF NEUROSURGERY
|
| Treatment protocol |
autopsy were used as non-epileptic controls and MTLE and FCD tissues were epileptic tissues
|
| Growth protocol |
Tissues resected from 2 autopsy , 3 MTLE and 4 FCD type II were used for isolating Genomic DNA
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted using genomic DNAextraction kit (Qiagen, Hilden, Germany) and Methylated DNA was immunoprecipitated using standard protocols with some modifications. Briefly, 4 µg MseI digested genomic DNA was immunoprecipitated with 10 µg of monoclonal mouse anti 5-methylcytidine antibody (New England Biolab, Pickering, Ontario and Abcam Inc.Cambridge, MA 02139) a negative control normal mouse IgG (Abcam, ab81032) in a final volume of 500 μl IP buffer (10 mM sodium phosphate (pH 7.0), 140 mM NaCl, 0.05% Triton X-100). We incubated the mixture with 40 μl of Dynabeads and M-280 sheep antibody to mouse IgG (Dynal Biotech) for 12 hr at 4°C and washed it seven times with 700 μl of IP buffer. We then treated the beads with proteinase K for 4 hr at 50°C and recovered the methylated DNA by phenol-chloroform extraction followed by ethanol precipitation. The immunoprecipitated DNA and a sample of input DNA were amplified using GenomePlex Complete Whole Genome Amplification (WGA) kit (Sigma-Aldrich, Saint Louis, Missouri 63103 USA). At each step DNA integrity was analyzed by agarose gel electrophoresis.
|
| Label |
cy5
|
| Label protocol |
2μg of DNA from the WGAamplified sample (MeDIP-WGA), along with their corresponding whole genomic DNA (input), were labeled usingthe SureTag DNA labeling kit (5190-3400, Agilent)
|
| |
|
| Channel 2 |
| Source name |
Mesial Temporal Lobe Epilepsy input DNA
|
| Organism |
Homo sapiens |
| Characteristics |
subject status: Mesial temporal epilepsy (MTLE) patient
age: 14
gender: MALE
tissue: brain
sample type: whole genomic DNA (input)
|
| Biomaterial provider |
DR. P. SARAT CHANDRA DEPT OF NEUROSURGERY
|
| Treatment protocol |
autopsy were used as non-epileptic controls and MTLE and FCD tissues were epileptic tissues
|
| Growth protocol |
Tissues resected from 2 autopsy , 3 MTLE and 4 FCD type II were used for isolating Genomic DNA
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted using genomic DNAextraction kit (Qiagen, Hilden, Germany) and Methylated DNA was immunoprecipitated using standard protocols with some modifications. Briefly, 4 µg MseI digested genomic DNA was immunoprecipitated with 10 µg of monoclonal mouse anti 5-methylcytidine antibody (New England Biolab, Pickering, Ontario and Abcam Inc.Cambridge, MA 02139) a negative control normal mouse IgG (Abcam, ab81032) in a final volume of 500 μl IP buffer (10 mM sodium phosphate (pH 7.0), 140 mM NaCl, 0.05% Triton X-100). We incubated the mixture with 40 μl of Dynabeads and M-280 sheep antibody to mouse IgG (Dynal Biotech) for 12 hr at 4°C and washed it seven times with 700 μl of IP buffer. We then treated the beads with proteinase K for 4 hr at 50°C and recovered the methylated DNA by phenol-chloroform extraction followed by ethanol precipitation. The immunoprecipitated DNA and a sample of input DNA were amplified using GenomePlex Complete Whole Genome Amplification (WGA) kit (Sigma-Aldrich, Saint Louis, Missouri 63103 USA). At each step DNA integrity was analyzed by agarose gel electrophoresis.
|
| Label |
Cy3
|
| Label protocol |
2μg of DNA from the WGAamplified sample (MeDIP-WGA), along with their corresponding whole genomic DNA (input), were labeled usingthe SureTag DNA labeling kit (5190-3400, Agilent)
|
| |
|
| |
| Hybridization protocol |
Labeled DNAs were hybridized to Human DNA methylation Microarray slides (G4495A, AMADID 023795) containing the annotated human 27,627 expanded CpG islands, 5081 UMR regions, 237,227 biological probes with median probe spacing of 97bp followingthe microarray manufacturer’s instructions.
|
| Scan protocol |
The slides were subsequently washed and scanned using Agilent scanner P/N G2565BA
|
| Description |
MTLE3
|
| Data processing |
Agilent Feature Extraction Software (v 8.5.1.1) was used for background subtraction and LOWESS normalization.
|
| |
|
| Submission date |
Mar 10, 2017 |
| Last update date |
Mar 10, 2018 |
| Contact name |
APARNA BANERJEE DIXIT |
| E-mail(s) |
aparnadixit@nbrc.ac.in
|
| Phone |
9810189197
|
| Organization name |
NATIONAL BRAIN RESERACH CENTRE
|
| Department |
A collaborative project between NBRC & AIIMS
|
| Lab |
Centre of Excellence for Epilepsy
|
| Street address |
Room No 7003, 7th Floor, Convegence Block, AIIMS, ANSARI, NAGAR
|
| City |
NEW DELHI |
| State/province |
AIIMS |
| ZIP/Postal code |
110029 |
| Country |
India |
| |
|
| Platform ID |
GPL19930 |
| Series (1) |
| GSE96067 |
CpG methylation analysis in Mesial temporal lobe epilepsy and Focal cortical dysplasia patients |
|
