Investigating the Effects of DNA Demethylation in Chemotherapy Resistance by ChIP-on-Chip Method

DNA Isolation and Immunoprecipitation
The experiments were conducted according to Agilent G4170-90012_Methylation_Protocol_v.2.0 in the laboratories of Selçuk University ILTEK. The genomic DNA isolation from MCF-7/S and MCF-7/Pac cells (1×106 cells from each) was performed using the genomic DNA isolation kit (QIAamp DNA mini kit, QIAGEN). The quality control procedure was performed by spectrophotometric measurements (OD260nm/OD280nm, Nanodrop-Thermo) and agarose gel electrophoresis. Genomic DNA (5 µg from each) was fragmented by ultrasonication by ultrasonicator (using micro probe, Heidolph) that was set output power to 70%. The DNA fragmentation was ensured by sonicating the DNA samples for 5 s, five cycles on ice by holding 5 s between each cycle intervals (to prevent heating and foaming). For immunoprecipitation procedure, Dynabeads Pan Mouse IgG magnetic beads were precipitated by magnetic stand (DynaMag-2 Magnet, Life Technologies), and the beads were bound to ChIP grade anti-5-Methyl Cytidine antibody (Abcam) by incubating in a rotator (VWR) at 4°C incubator. The fragmented DNA samples were then hybridized with the magnetic bead bound 5-methylcytidine antibody in immunoprecipitation buffer (Triton X, yeast t-RNA, PBS) to let the binding of methylated DNA fragments on to the magnetic antibodies (in rotator, overnight at 4°C incubator). Finally, the methylated DNA fragments were sorted on magnetic stand. The methylated DNA samples and reference DNA samples (that were not immunoprecipitated) were eluted by elution in TE and SDS solution. DNA fragments were extracted through phenol-chloroform extraction method by use of MaXtract High Density tubes (QIAGEN). The aqueous phase was collected, and DNA was precipitated by adding the precipitation solution that constitutes NaCl (200 mM), glycogen 20 ?g/mL, and pure ethanol. DNA was precipitated by incubating the mixture at 4°C and centrifugation at 12.000 g for 3 min (Hettich). Finally, the DNA pellets were air dried. The quality of eluted methylated DNA and reference DNA samples of MCF-7/Pac and MCF- 7/S were measured by Nanodrop (Thermo).

Fluorescent Labeling of DNA Samples
Methylated and reference DNA fragments were labeled using the SureTag DNA Labeling Kit (Agilent). Briefly, DNA samples were mixed with random primers and incubated at 95°C for 3 min on heating-cooling dry block (Biosan). While DNA samples were denatured, they were labeled by Exo Klenow enzyme, dNTPs. and fluorescently labeled dUTPs (Cy-3 and Cy-5 labeled) at 37°C for 2 h in PCR machine (Biorad). After the reaction was stopped at 65°C, labeled DNA fragments were purified by purification columns, and fluorescence binding success was determined by spectrophotometric measurements (Nanodrop, Thermo). Specific activity and labeled DNA amounts were calculated using the following formulas:

Specific activity=pmol dye per µL of dye/µg per µL DNA Yield (µg)=DNA concentration (ng/µL)×sample volume (µL)/1000 ng/?g

Hybridization of Labeled DNA on Arrays
Since DNA labeling quality was good (yield >2.5 µg and appropriate specific activity), the labeled DNA fragments were hybridized on the array platforms (Human DNA Methylation Microarray, 1×244K (HD)). Briefly, the hybridization master mix was prepared for four samples (duplicates for each cell line). Hybridization master mix constituted Human Cot-1 DNA for internal control, CGH (chromosomal genomic hybridization) blocking agent, HI-RPM hybridization buffer, and deionized formamide. Hybridization master mix (420 µL for 1-pack microarray format) was combined with Cy3- and Cy5-labeled DNA samples (40 µL each) in one tube for each cell line. Two replicates of hybridization mixtures were prepared for MCF-7/S and MCF-7/Pac cell lines and incubated at 95°C for 3 min and 37°C for 30 min in thermal cycler (Biorad). The hybridization solution should be kept at 37°C until it is loaded on to the array slides. Hybridization solution was slowly dispensed on to the gasket slide carefully preventing the overflow of the solution out of the gasket chamber. Then the printed microarray slide (Agilent, Unrestricted Amadid Chip-on-Chip 1×244K) was put on to the gasket slide (barcode number should be outside). Finally, the microarray slide was assembled in the slide chamber. The hybridization oven was set to 67°C, and the chambers were placed in to the array holders in a balanced way. The hybridization proceeded for 40 h at 20 rpm rotation speed. Two repeats were performed for each group (MCF-7/S and MCF-7/ Pac), (n=2).

Washing and Scanning
The washing equipments were washed with acetonitrile and ultrapure water previously. ChIP-on-chip wash buffer 2 was prewarmed in a coplin jar before disassembling the slide chambers (37°C). The slides were disassembled in ChIP-on-chip wash buffer 1, in to a separate coplin jar. Then the slides were put into another jar that contains wash buffer 1 for 5 min at room temperature (a magnetic bead was put at the basement of the jar to provide continuous stirring by magnetic stirrer). Arrays were immediately air dried, and ozone covers were put on to prevent adverse effects of the ozone on to the slides. During the whole procedure, slides should be kept by forceps. Sure Scan Microarray Scanner (Agilent), which was in the laboratory of Selçuk University ILTEK, was turned on to warm up the lasers. The G4900DA SureScan Microarray Scanner System Microarray Scan Control Software 9.1 (Agilent) was run, and Protocol Agilent HD-CGH was selected. The slides were immediately inserted into the scanner as the scanner was ready to scan the arrays. The machine recognized the barcode of the array. The green and red lasers excited the fluorescent dyes, and then photomultiplier tube detector detected the data. After scan protocol was completed, the Feature Extraction software v11.0.1.1 was operated for quality control (QC). The QC reports were generated using the grid Human DNA Methylation Microarray 244k- 023795-D-F-20111018.

Advanced Analysis and Statistics
The scan data of each cells were analyzed by Genomic Workbench ver6.0 (Agilent) program. Z-score algorithm was used for calculations. Gaussians were fitted to the bimodal log ratio distribution. Significant changes were determined between the groups (MCF- 7/S and MCF-7/Pac) by Student"s t-test (p<0.05). Finally, the significant results were listed, and the demethylation levels were calculated by calculating the fold change values and presented as the logarithm of the fold change ratios (MCF-7/Pac/MCF-7).