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Steven Roberts added methyl miner and array description
about 9 years ago
Commit id: 8f7807184a7327097d18553b9984925ac28a2d7d
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#methods-array
Genomic DNA was
extracted isolated using
DNAzol (Molecular Research Center) from mantle tissue taken prior to and post heat shock (three oysters, six samples) Methylation enrichment performed using the MethylMiner Kit (Invitrogen) following the manufacturer’s instructions. Specifically, DNA was sheared by sonication on a Covaris S2 (Covaris) (parameters: 10 cycles at 60 seconds each, duty cycle of 10%, intensity of 5, 100 cycles/burst). Sheared DNA was used as input DNA and incubated with MBD-Biotin Protein coupled to M-280 Streptavidin Dynabeads following the manufacturer’s instructions (MethylMiner (Invitrogen)). Enriched, methylated DNA was eluted from the bead complex with 1M NaCl and purified by ethanol precipitation. DNA was further purified using PCR purification columns (Qiagen) prior to labeling.
A custom DNA tiling array containing 697,753 probes covering 9158 full-length C. gigas genes including 2kb upstream of the start site was used. Probes were designed using an interval size of 100bp and a window size of 25bp.
> Include how comparisons were made..
Samples were labeled using the NimbleGen Dual-Color DNA Labeling Kit and the arrays were processed according to the manufacturer’s recommendations (Roche NimbleGen, Madison, Wisconsin) and imaged at 5um using a GenePix 4000B microarray scanner (Molecular Devices, Sunnyvale, CA).
Raw data from both the Cy3 and Cy5 channels were .........
