After surgical excision, tissue samples for nucleic acids extraction were immediately sliced into aliquots of about 100 mg, snap-frozen in liquid nitrogen and stored at -80°C until use. Aliquots for DNA extraction were homogenized in 2ml of chilled NaCl 0.9% w/v; cell lysis was achieved by using Nonidet P40 (Sigma-Aldrich, St. Louis, MO, USA) 0.1% and lysis solution (NaCl 100 mM, EDTA 25 mM, SDS 1.6 %, pH 8). Samples were treated with proteinase K/RNase and DNA extracted with a standard phenol/chloroform procedure. The methylated DNA immunoprecipitation (MeDIP) was performed with MeDIP kit™ by Diagenode (Liège, Belgium). IP and INPUT samples were amplified by GenomePlex Complete Genome Amplification (WGA) kit (Sigma-Aldrich, St. Louis, MO, USA) following the producer’s protocol.
Label
Cy5
Label protocol
One and a half μg of IP and 1.5 μg INPUT samples were labelled with Cy5 and Cy3 respectively by Dual-Color DNA Labeling Kit (NimbleGen-Roche, Madison, WI, USA).
After surgical excision, tissue samples for nucleic acids extraction were immediately sliced into aliquots of about 100 mg, snap-frozen in liquid nitrogen and stored at -80°C until use. Aliquots for DNA extraction were homogenized in 2ml of chilled NaCl 0.9% w/v; cell lysis was achieved by using Nonidet P40 (Sigma-Aldrich, St. Louis, MO, USA) 0.1% and lysis solution (NaCl 100 mM, EDTA 25 mM, SDS 1.6 %, pH 8). Samples were treated with proteinase K/RNase and DNA extracted with a standard phenol/chloroform procedure. The methylated DNA immunoprecipitation (MeDIP) was performed with MeDIP kit™ by Diagenode (Liège, Belgium). IP and INPUT samples were amplified by GenomePlex Complete Genome Amplification (WGA) kit (Sigma-Aldrich, St. Louis, MO, USA) following the producer’s protocol.
Label
Cy3
Label protocol
One and a half μg of IP and 1.5 μg INPUT samples were labelled with Cy5 and Cy3 respectively by Dual-Color DNA Labeling Kit (NimbleGen-Roche, Madison, WI, USA).
Hybridization protocol
Samples were hybridized on the Human DNA Methylation 3x720K CpG Island Plus RefSeq Promoter Array (NimbleGen-Roche, Madison, WI, USA) following manufacturer instructions
Scan protocol
Microarrays were scanned with Axon GenePix 4400A microarray scanner setting laser power to 100%. PMT gain was set in order to obtain about 1e-5 normalized counts at 65.000 intensity level for both channels.
Description
MeDIP-chip non neoplastic liver tissue
Data processing
Data were quantile normalized and logged fold change of immunoprecipitated sample (IP) over control (INPUT) sample was calculated
