|
| Status |
Public on Aug 13, 2013 |
| Title |
S5_4yrs_F_naive |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
MIRA-enriched DNA
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: methylated DNA fraction
subject: 5
age: 4
gender: F
cell type: tonsils
cell type: naive
|
| Treatment protocol |
FACS purified
|
| Growth protocol |
isolated ex vivo from tonsil
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was purified using Allprep DNA/RNA/protein columns (Qiagen). Purified genomic DNA was sonicated using a Bioruptor (Diagenode) to generate 200-500 bp fragments. Methylated CpG DNA fragments were enriched from 200 ng of sonicated DNA using the MethylCollector Ultra Kit (Actif Motif) following the manufacturer’s protocol. 20 ng of input and enriched DNA fractions were amplified twice with WGA2 (Sigma).
|
| Label |
cy5
|
| Label protocol |
Amplified input and methylated CpG DNA fragments labeled with Cy3 and Cy5 random nonomers (Trilink Biotechnologies) following NimbleGen’s protocol.
|
| |
|
| Channel 2 |
| Source name |
Input DNA
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: input DNA
subject: 5
age: 4
gender: F
cell type: naive
|
| Treatment protocol |
FACS purified
|
| Growth protocol |
isolated ex vivo from tonsil
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was purified using Allprep DNA/RNA/protein columns (Qiagen). Purified genomic DNA was sonicated using a Bioruptor (Diagenode) to generate 200-500 bp fragments. Methylated CpG DNA fragments were enriched from 200 ng of sonicated DNA using the MethylCollector Ultra Kit (Actif Motif) following the manufacturer’s protocol. 20 ng of input and enriched DNA fractions were amplified twice with WGA2 (Sigma).
|
| Label |
cy3
|
| Label protocol |
Amplified input and methylated CpG DNA fragments labeled with Cy3 and Cy5 random nonomers (Trilink Biotechnologies) following NimbleGen’s protocol.
|
| |
|
| |
| Hybridization protocol |
Samples were hybridized onto a NimbleGen 2.1M Deluxe Human Promoter Array following NimbleGen’s protocol.
|
| Scan protocol |
The microarray slides were scanned using the Agilent G2565BA DNA Microarray Scanner. Images were processed using the NimbleScan software.
|
| Description |
This is naive MIRA-chip sample from subject 5
|
| Data processing |
The log2(Cy5) values were normalized using Lowess curve fitting conditioning on GC content as described in BMC Bioinformatics 2009, 10:173. These signal values were further quantile normalized to adjust for between sample variations.
The GC Lowess method referred in this fields utilizes both 'log2(Cy3)' and 'log2(Cy5)' values as following:
Classify probes into bins according to CG content (proportion of number of C and G nucleotides in probe sequence)
For each CG content bin (k), we fit lowess regression to predict log2(Cy5)_k value using log2(Cy3)_k value
Compute normalized value = sigma_bar *(observed log2(Cy5)_k - predicted log2(Cy5)_k)/sigma_k, where sigma_k is mean absolute deviation of (observed log2(Cy5)_k - predicted log2(Cy5)_k) and sigma_bar is geometric mean of all sigma_k's.
This normalization corrects for both dye bias and CG bias simultaneously.
|
| |
|
| Submission date |
Oct 25, 2010 |
| Last update date |
Aug 13, 2013 |
| Contact name |
Deepak Mav |
| Organization name |
Sciome LLC
|
| Street address |
2 Davis Drive
|
| City |
Research Triangle Park |
| State/province |
NC |
| ZIP/Postal code |
27709 |
| Country |
USA |
| |
|
| Platform ID |
GPL10671 |
| Series (2) |
| GSE24918 |
B lymphocyte activation induces DNA methylation reprogramming and establishes a common epigenetic signature in memory B and plasma cell compartments [DNA methylation profiling] |
| GSE24919 |
B lymphocyte activation induces DNA methylation reprogramming and establishes a common epigenetic signature in memory B and plasma cell compartments |
|
