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Sample GSM605951

Query DataSets for GSM605951

Status

Public on Oct 08, 2010

Title

Prec1_MeDNA_Input1

Sample type

genomic

Source name

PrEC

Organism

Homo sapiens

Characteristics

cell type: cell line
cell source: PrEC
treatment group: none

Treatment protocol

none

Growth protocol

37 deg C with 5% CO2

Extracted molecule

genomic DNA

Extraction protocol

The MeDIP assay was performed on 4 µg of sonicated genomic DNA (300-500 bp) in 1x IP buffer (10 mM sodium phosphate pH 7.0, 140 mM NaCl and 0.05% Triton X-100). Ten µg anti-5-methylcytosine mouse monoclonal antibody (Calbiochem clone 162 33 D3 Cat No. NA81) was incubated overnight in 500 µl 1x IP buffer and the DNA/antibody complexes were collected with 80 µl Protein A/G PLUS agarose beads (Santa Cruz sc-2003). The beads were washed 3 times with 1x IP buffer at 4C and twice with 1 ml TE buffer at room temperature. The immune complexes were eluted with freshly prepared 1% SDS, 0.1 M NaHCO3, and the DNA was purified by phenol/chloroform extraction, ethanol precipitation and resuspended in 30 µl H2O. Immunoprecipitated and Input DNA was amplified with 14 cycles of whole genome amplification (WGA2 kit, Sigma) according to manufacturers instructions. MBDCap: The MethylMiner™ Methylated DNA Enrichment Kit (Invitrogen) was used to isolate methylated DNA. 1 µg of genomic DNA was sonicated to 100-500 bps. 3.5 µg (7 µl) of MBD-Biotin Protein were coupled to 10 µl of Dynabeads M-280 Streptavidin according to the manufacturer’s instructions. The MBD-magnetic beads conjugates were washed 3 times and resuspended in 1 volume of 1 x Bind/Wash buffer. The capture reaction was performed by adding 1 µg sonicated DNA to the MBD-magnetic beads on a rotating mixer for 1 hour at room temperature. All capture reactions were done in duplicate. The beads were washed three times with 1 x Bind/Wash buffer. The methylated DNA was eluted in one of two ways: (1) as a single fraction with a High Salt Elution Buffer (2000mM NaCl), denoted MBD-SF, or (2) as distinct subpopulations based on the degree of methylation using an increasing NaCl concentration of the elution buffer, from 200mM to 2000mM in a stepwise gradient (Elution 1- 200mM, Elution 2 – 350mM, Elution 3 – 450mM, Elution 4 -600mM, Elution 5 -1000mM and Elution 6 – 2000mM). Each fraction was concentrated by ethanol precipitation using 1 µl glycogen (20 µg/µl), 1/10th volume of 3M sodium acetate, pH 5.2 and two sample volumes of 100% ethanol and resuspended in 60 µl H20.

Label

biotin

Label protocol

Samples (7.5μg of DNA) were enzymatically fragmented and labeled using the WT Labeling Kit (Affymetrix) to add biotinylated compound to the ends of the fragments

Hybridization protocol

As per standard Affymetrix protocol for the Human Promoter 1.0R tiling arrays

Scan protocol

As per standard Affymetrix protocol for the Human Promoter 1.0R tiling arrays

Data processing

All analysis was performed with UCSC hg18 version of the human genome. The data was processed using the MAT (Johnson WE et al PNAS 2006) implementation available in the R/aroma.affymetrix package, as well as the R/Repitools package (Statham AL et al Bioinformatics 2010). Untargeted differentially methylated regions (DMRs) between LNCaP and PrEC cells were determined using the regionStats() procedure in the Repitools package. DMRs at transcription start sites were determined using the blocksStats() procedure in the Repitools package.
The Series supplemental archive 'GSE24546_MeDIP-chip_results.tar' contains the differential methylation calls from the different methods (regionStats_d_ELUTION_5.txt, regionStats_d_MEDIP.txt, regionStats_d_SF.txt) as well as the complete RMA normalized inputs (inputsRMA.txt).

Submission date

Oct 07, 2010

Last update date

Oct 08, 2010

Contact name

Mark Robinson

E-mail(s)

[email protected]

Organization name

University of Zuerich

Department

Institute of Molecular Life Sciences