|
| Status |
Public on Aug 10, 2009 |
| Title |
hypomethylated pools Replicate 1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
gDNA from BMM; MCIp treated, hypomethylated pool
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
gender: male
Age: 8-12 weeks
Tissue: bonemarrow
|
| Growth protocol |
Bone marrow progenitor cells were flushed from femur and tibia of C57BL/6 & BALB/c mice with TBS using 27 gauge needle attached to a 5 ml syringe from both directions, centrifuged at 300 g, for 8 min at 4°C counted and seeded at a concentration of 5*10E5 cells/ml culture medium (90 % 1640 RPMI, 10 % fetal calf serum (FCS) supplied with 1 mM sodium pyruvate (Gibco), 2 mM L-Glutamine (Biochrome), 2 ml MEM Non-essential amino acids (Gibco), MEM Vitamines (Gibco), 50 U/ml Penicillin/Streptomycin (Gibco), 50 µM 2-Mercaptoethanol (Gibco)and 200 ng/ml rCSF (Cetus)) in bacteriological square plates in an humified incubator at 37°C and 5 % CO2. After 5 days the medium was changed and cells were harvested on day 6 and replated at a density of 10*10E6 cells/10 ml media in 10 cm tissue culture dish. Cells were harvested on day 7.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted from BMM's using Qiagen Blood & Cell Culture DNA Kit and Genomic-tip 100/G according to the manufacturer's instructions.
|
| Label |
Alexa Fluor 5
|
| Label protocol |
MCIp treated genomic DNA pools were labeled using the BioPrime Total Genomic Labeling System (Invitrogen; Version A, 12 February 2007) according to the manufacturer's intruction.
|
| |
|
| Channel 2 |
| Source name |
gDNA from BMM; MCIp treated, hypomethylated pool
|
| Organism |
Mus musculus |
| Characteristics |
Strain: BALB/c
gender: male
Age: 8-12 weeks
Tissue: bonemarrow
|
| Growth protocol |
Bone marrow progenitor cells were flushed from femur and tibia of C57BL/6 & BALB/c mice with TBS using 27 gauge needle attached to a 5 ml syringe from both directions, centrifuged at 300 g, for 8 min at 4°C counted and seeded at a concentration of 5*10E5 cells/ml culture medium (90 % 1640 RPMI, 10 % fetal calf serum (FCS) supplied with 1 mM sodium pyruvate (Gibco), 2 mM L-Glutamine (Biochrome), 2 ml MEM Non-essential amino acids (Gibco), MEM Vitamines (Gibco), 50 U/ml Penicillin/Streptomycin (Gibco), 50 µM 2-Mercaptoethanol (Gibco)and 200 ng/ml rCSF (Cetus)) in bacteriological square plates in an humified incubator at 37°C and 5 % CO2. After 5 days the medium was changed and cells were harvested on day 6 and replated at a density of 10*10E6 cells/10 ml media in 10 cm tissue culture dish. Cells were harvested on day 7.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted from BMM's using Qiagen Blood & Cell Culture DNA Kit and Genomic-tip 100/G according to the manufacturer's instructions.
|
| Label |
Alexa Fluor 3
|
| Label protocol |
MCIp treated genomic DNA pools were labeled using the BioPrime Total Genomic Labeling System (Invitrogen; Version A, 12 February 2007) according to the manufacturer's intruction.
|
| |
|
| |
| Hybridization protocol |
Samples were hybridized using a stringent protocol. The differentially labelled genomic pools (ether hypo or hyper) were combined and supplemented with 50 µg mouse Cot-1 DNA (Invitrogen), 52 µl of Agilent blocking agent (10-fold) (Agilent), 15% Deionized formamide (Sigma) and 250 µl Agilent hybridization buffer (2-fold) as supplied in the Agilent oligo aCGH Hybridization kit. The samples are heated to 95° for 3 min, mixed and subsequently incubated at 37°C for 30 min. Hybridization was then carried out 67°C for 40 h using an SureHyb chamber and an Agilent hybridization oven. Slides were washed in Wash I (6xSSPE, 0.005% N-lauroylsarcosine) at room temperature for 5 min and in prewarmed Wash II (37°C, 0.06xSSPE) for an additional 5 min. Afterwards slides were dried using acetonitrile within an ozone free facility.
|
| Scan protocol |
Scanned on an Agilent scanner at 5 µm resolution.
Images were quantified using Agilent Feature Extraction Software (version 9.5.1).
|
| Description |
Biological replicate 1 of 2. Corresponding hypermethylated genome pools were utilized for hyper-rep1
|
| Data processing |
Linear normalized, background subtracted VALUE data obtained from log10 of processed Red signal/processed Green signal. Agilent software was used. Processed signal intensities were further normalized using GC-dependent regression and imported into Microsoft Office Excel 2007 for further analysis.
|
| |
|
| Submission date |
Jan 16, 2009 |
| Last update date |
Aug 10, 2009 |
| Contact name |
Elmar Schilling |
| E-mail(s) |
elmar.schilling@klinik.uni-regensburg.de
|
| Phone |
**49 941-944-5591
|
| Organization name |
University Hospital Regensburg
|
| Street address |
Franz-Josef-Strauss Allee 11
|
| City |
Regensburg |
| ZIP/Postal code |
93047 |
| Country |
Germany |
| |
|
| Platform ID |
GPL8085 |
| Series (1) |
| GSE14463 |
Methylation profiling of differentially expressed regions between C57BL/6 and BALB/c in bone marrow derived macrophages |
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