|
| Status |
Public on Oct 23, 2014 |
| Title |
6L mDNA |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
MIRA DNA_primary skin fibroblast_late
|
| Organism |
Homo sapiens |
| Characteristics |
donor age (yrs): 54
cell type: primary skin fibroblast
time point: late timepoint (L)
chip antibody: MIRA
|
| Growth protocol |
Primary Skin Fibroblast cells were grown with culture medium (DMEM supplemented with 6% fetal bovine serum) for 2 -3 passages.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Primary Embryonic Fibroblast cells were grown in a 150 mm dish containing 25 ml of culture medium (DMEM supplemented with 6% fetal bovine serum (FBS) to about 5x107 cells). Fresh formaldehyde was added directly into media to a final concentration of 1% and incubated at RT for exactly 2 minutes on an orbiting platform. Cross-linking reaction was stopped by adding 2.6 ml of 1.25 M glycine solution. Media was removed and cells were washed twice with 20 ml ice-cold PBS without Ca and Mg. Cells were pelleted by centrifugation for 3 minutes at 250g at 4°C. For sonification, cells were resuspended from each 150 mm dish in 350 µl ChIP Lysis Buffer (1% SDS, 10 mM EDTA and 50 mM Tris-HCl, pH 8.1) containing protease inhibitors (complete protease inhibitor cocktail tablets, Roche applied science, Cat.# 1697498). Sonication was carried out in 1.7 ml Eppendorf tubes using Branson Sonifer with a micro tip. Sonication condition were 10 times at 15sec on, 45sec off, at output 25%. Sheared chromatin was centrifuged at 14,000 rpm for 15 minutes at 4C. For Methylated-CpG island recovery assay (MIRA), the methylated faction of sonicated genomic DNA from human skin fibroblast cells was captured using recombinant MBD3L1 and MBD2b proteins. Ligation-mediated PCR (LM-PCR) was performed to amplify MIRA-enriched DNA.
|
| Label |
Cy5
|
| Label protocol |
According to standard NimbleGen Protocol
|
| |
|
| Channel 2 |
| Source name |
Input DNA from primary skin fibroblast, early timepoint
|
| Organism |
Homo sapiens |
| Characteristics |
donor age (yrs): 54
cell type: primary skin fibroblast
time point: early timepoint (E)
chip antibody: none, input DNA
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Primary Embryonic Fibroblast cells were grown in a 150 mm dish containing 25 ml of culture medium (DMEM supplemented with 6% fetal bovine serum (FBS) to about 5x107 cells). Fresh formaldehyde was added directly into media to a final concentration of 1% and incubated at RT for exactly 2 minutes on an orbiting platform. Cross-linking reaction was stopped by adding 2.6 ml of 1.25 M glycine solution. Media was removed and cells were washed twice with 20 ml ice-cold PBS without Ca and Mg. Cells were pelleted by centrifugation for 3 minutes at 250g at 4°C. For sonification, cells were resuspended from each 150 mm dish in 350 µl ChIP Lysis Buffer (1% SDS, 10 mM EDTA and 50 mM Tris-HCl, pH 8.1) containing protease inhibitors (complete protease inhibitor cocktail tablets, Roche applied science, Cat.# 1697498). Sonication was carried out in 1.7 ml Eppendorf tubes using Branson Sonifer with a micro tip. Sonication condition were 10 times at 15sec on, 45sec off, at output 25%. Sheared chromatin was centrifuged at 14,000 rpm for 15 minutes at 4C. For Methylated-CpG island recovery assay (MIRA), the methylated faction of sonicated genomic DNA from human skin fibroblast cells was captured using recombinant MBD3L1 and MBD2b proteins. Ligation-mediated PCR (LM-PCR) was performed to amplify MIRA-enriched DNA.
|
| Label |
Cy3
|
| Label protocol |
According to standard NimbleGen Protocol
|
| |
|
| |
| Hybridization protocol |
According to standard NimbleGen Protocol
|
| Scan protocol |
Follow NimbleScan's default using Agilent Scanner
|
| Description |
MIRA-chip primary skin fibroblasts, late timepoint mDNA
Sample 50
|
| Data processing |
Samples were quantile-normalized and arrays were normalized by Nimblegen's recommended method, tukey-biweight.
|
| |
|
| Submission date |
Oct 21, 2013 |
| Last update date |
Oct 23, 2014 |
| Contact name |
Marc Jung |
| E-mail(s) |
[email protected]
|
| Organization name |
City Of Hope
|
| Department |
Biology
|
| Lab |
Gerd Pfeifer
|
| Street address |
1500 East Duarte Street
|
| City |
Duarte |
| State/province |
CA |
| ZIP/Postal code |
91010 |
| Country |
USA |
| |
|
| Platform ID |
GPL17148 |
| Series (2) |
| GSE51517 |
Chip-chip and MIRA-chip from primary skin fibroblasts, derived of matched pairs of early and late donor age |
| GSE51519 |
Aging signatures developed from a longitudinal study design are dominated by reduced transcription of genes involved in protein synthesis. |
|
