|
| Status |
Public on Dec 11, 2013 |
| Title |
T cell (CD3 positive)_medip_control_aggressivity.19 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
T cell (CD3 positive)_meDIP_control_aggressivity
|
| Organism |
Homo sapiens |
| Characteristics |
gender: female
cell type: T cell (CD3 positive)
state: control aggressivity trajectory
antibody: anti-5 methylcytosine antibody (Calbiochem, Cat. # NA81, Lot: D00048001)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
20 ml of blood were drawn in EDTA coated-tubes for each subjects. PBMC (whole mononuclear cells from peripheral blood) and T cell isolation procedures were adapted from Current Protocols in Immunology (1997, sections 7.1 and 7.5.1-7.5.11). Briefly, PBMC isolation was done by centrifugation with Ficoll-Paque (GE healthcare) and washed twice with HBSS (Hanks balanced salt solution, GIBCO). T cells were isolated from the PBMCs by immunomagnetic positive selection using CD3 dynabeads (Dynal). The beads were washed 3 times and incubated with the PBMCs for 45 min on a rotator at 4C. Coated CD3+ cells with the dynabeads were isolated using a strong magnet (Steam Cell Technology) and washed 5 times with PBS/FBS. CD3+ cells coated with the dynabeads were then frozen at -80C until DNA extraction. T cells DNA was extracted with Wizard Genomic DNA Purification kit (Promega) following the manufacturer's protocol. For MeDIP analysis, 2 ug of the T cells DNA were sonicated and methylated DNA was immunoprecipitated with 10ug of anti-5methylCytosine (Calbiochem, Cat. # NA81, Lot: D00048001). The input and bound fractions were then amplified in triplicate using the Whole Genome Amplification kit (Sigma).
|
| Label |
Cy5
|
| Label protocol |
The input and bound fractions were amplified using Whole Genome Amplification kit (Sigma) and labeled for microarray hybridization with either Cy3-dUTP or Cy5-dUTP (Perkin Elmer) respectively using the CGH labeling kit (Invitrogen) following the manufacturer's instructions. 2ug of each of the input and bound fractions were labeled.
|
| |
|
| Channel 2 |
| Source name |
T cell (CD3 positive)_input_DNA_control_aggressivity
|
| Organism |
Homo sapiens |
| Characteristics |
gender: female
cell type: T cell (CD3 positive)
state: control aggressivity trajectory
antibody: none
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
20 ml of blood were drawn in EDTA coated-tubes for each subjects. PBMC (whole mononuclear cells from peripheral blood) and T cell isolation procedures were adapted from Current Protocols in Immunology (1997, sections 7.1 and 7.5.1-7.5.11). Briefly, PBMC isolation was done by centrifugation with Ficoll-Paque (GE healthcare) and washed twice with HBSS (Hanks balanced salt solution, GIBCO). T cells were isolated from the PBMCs by immunomagnetic positive selection using CD3 dynabeads (Dynal). The beads were washed 3 times and incubated with the PBMCs for 45 min on a rotator at 4C. Coated CD3+ cells with the dynabeads were isolated using a strong magnet (Steam Cell Technology) and washed 5 times with PBS/FBS. CD3+ cells coated with the dynabeads were then frozen at -80C until DNA extraction. T cells DNA was extracted with Wizard Genomic DNA Purification kit (Promega) following the manufacturer's protocol. For MeDIP analysis, 2 ug of the T cells DNA were sonicated and methylated DNA was immunoprecipitated with 10ug of anti-5methylCytosine (Calbiochem, Cat. # NA81, Lot: D00048001). The input and bound fractions were then amplified in triplicate using the Whole Genome Amplification kit (Sigma).
|
| Label |
Cy3
|
| Label protocol |
The input and bound fractions were amplified using Whole Genome Amplification kit (Sigma) and labeled for microarray hybridization with either Cy3-dUTP or Cy5-dUTP (Perkin Elmer) respectively using the CGH labeling kit (Invitrogen) following the manufacturer's instructions. 2ug of each of the input and bound fractions were labeled.
|
| |
|
| |
| Hybridization protocol |
All the steps of hybridization, washing, and scanning were performed following the Agilent protocol for ChIP-on-chip analysis. Cot-1 DNA was added to the hybridization mix to minimize cross-hybridization between repetitive element.
|
| Scan protocol |
Scanning was performed using the Agilent Technologies Scanner G2505B.
|
| Description |
control aggressivity trajectory
control aggressivity trajectory
comparison of T cell DNA methylation in individuals with different aggressivity trajectories
|
| Data processing |
After microarray scanning, probe intensities were extracted from scan images using Agilent's Feature Extraction 9.5.3 Image Analysis Software. Quality control involved visual inspection of microarray scan images, scanning software reports and generation of MvA plots (i.e. plots of log(Cy5/Cy3) vs log(Cy5 x Cy3)) to identify microarrays with dye biases or low signal. Microarrays were normalized using background subtraction followed by quantile normalization of log2-ratios of bound (Cy5) and input (Cy3) channel intensities.
|
| |
|
| Submission date |
Dec 10, 2013 |
| Last update date |
Dec 11, 2013 |
| Contact name |
Moshe Szyf |
| E-mail(s) |
moshe.szyf@mcgill.ca
|
| Organization name |
McGill University
|
| Department |
Pharmacology
|
| Street address |
McIntyre Medical Building, 3655 Promenade Sir William Osler, Room 1309
|
| City |
Montreal |
| State/province |
Quebec |
| ZIP/Postal code |
H3G 1Y6 |
| Country |
Canada |
| |
|
| Platform ID |
GPL18047 |
| Series (2) |
| GSE53192 |
DNA Methylation Signature of Childhood Chronic Physical Aggression in T Cells of Both Men and Women (Agilent) |
| GSE53193 |
DNA Methylation Signature of Childhood Chronic Physical Aggression in T Cells of Both Men and Women |
|
