cell type: A549 human epithelial lung carcinoma cells treatment: 2 µM of sodium arsenite time: 2 weeks sample type: input DNA
Extracted molecule
genomic DNA
Extraction protocol
Cells were washed twice with Hank's Buffered Salt Solution (Invitrogen) and trypsinized. Cell pellets were resuspended in 500 µl digestion buffer (1 mM EDTA; 50 mM Tris–HCl, pH 8.0; 5% SDS) and proteinase K (1 mg/ml) (Ambion) was added. After incubation of 1 hour at 55 °C, the proteinase K was inactivated at 80 °C. RNAse A (400 µg/ml) (Qiagen) treatment was performed for 1 hour at 37 °C. An equal amount of phenol-chloroform-isoamylalcohol (PCI; 25:24:1) (Sigma) was added and shaked manually for 5 min. After centrifugation, the upper phase was again treated with PCI until no protein was visible at the interphase. The upper phase was precipitated with 50 µl 3M NaAc pH 5.6 and 1250 µl cold 100% ethanol. The DNA pellet was washed with cold 70% ETOH, dissolved in 50µl nuclease free water and quantified spectrophotometrically using the NanoDrop 1000 (Thermo Scientific, Waltham, MA). The total amount was at least 10 µg DNA, the 260/280 ratio laid between 1.7-1.9, and the 260/230 was higher than 1.6.
Label
Cy3
Label protocol
Genomic DNAs were sonicated to obtain fragments ranging from 200 bp to 1000 bp, cleaned up using silica columns (Zymo Research) and eluted in TE buffer. 4.4 µg of each sonicated sample was dissolved in a total volume of 495 µL TE buffer. Ten percent of such prepared mixtures was kept as Input sample (Input) at 4°C, while the remaining was immunoprecipitated with 12 µL of a monoclonal antibody against 5’-methylcytidine (Eurogentec) as described by Weber et al (Weber M, JJ Davies, D Wittig, EJ Oakeley, M Haase, WL Lam and D Schubeler. 2005. Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nat Genet 37: 853-862.). Immunoprecipitated DNAs (MeDIP) were purified using silica columns (Zymo Research) and eluted in TE buffer. Forty ng of both Input and MeDIP samples were whole genome amplified (WGA) using the WGA2 kit (Sigma Aldrich) following the manufacturer’s instruction, but omitting the fragmentation step. WGA reactions were cleaned up using silica columns (Sigma Aldrich) and eluted in water. Labeling and hybridization of arrays was performed according to the manufactures’ protocol. In brief, 1 µg of Input DNA and 1 µg of MeDIP DNA were labeled with Cy3 and Cy5 respectively by random priming using the Dual Color DNA labeling kit (Roche NimbleGen). The labeled samples were precipitated using isopropanol and quantified spectrophotometrically using the NanoDrop 1000.
Channel 2
Source name
medip DNA from A549 cells exposed to 2 µM of sodium arsenite for 2 weeks replicate 2
cell type: A549 human epithelial lung carcinoma cells treatment: 2 µM of sodium arsenite time: 2 weeks antibody: monoclonal antibody against 5’-methylcytidine (Eurogentec, Weber et al (2005), PMID: 16007088) sample type: immunoprecipitated methyl-DNA (MeDIP)
Extracted molecule
genomic DNA
Extraction protocol
Cells were washed twice with Hank's Buffered Salt Solution (Invitrogen) and trypsinized. Cell pellets were resuspended in 500 µl digestion buffer (1 mM EDTA; 50 mM Tris–HCl, pH 8.0; 5% SDS) and proteinase K (1 mg/ml) (Ambion) was added. After incubation of 1 hour at 55 °C, the proteinase K was inactivated at 80 °C. RNAse A (400 µg/ml) (Qiagen) treatment was performed for 1 hour at 37 °C. An equal amount of phenol-chloroform-isoamylalcohol (PCI; 25:24:1) (Sigma) was added and shaked manually for 5 min. After centrifugation, the upper phase was again treated with PCI until no protein was visible at the interphase. The upper phase was precipitated with 50 µl 3M NaAc pH 5.6 and 1250 µl cold 100% ethanol. The DNA pellet was washed with cold 70% ETOH, dissolved in 50µl nuclease free water and quantified spectrophotometrically using the NanoDrop 1000 (Thermo Scientific, Waltham, MA). The total amount was at least 10 µg DNA, the 260/280 ratio laid between 1.7-1.9, and the 260/230 was higher than 1.6.
Label
Cy5
Label protocol
Genomic DNAs were sonicated to obtain fragments ranging from 200 bp to 1000 bp, cleaned up using silica columns (Zymo Research) and eluted in TE buffer. 4.4 µg of each sonicated sample was dissolved in a total volume of 495 µL TE buffer. Ten percent of such prepared mixtures was kept as Input sample (Input) at 4°C, while the remaining was immunoprecipitated with 12 µL of a monoclonal antibody against 5’-methylcytidine (Eurogentec) as described by Weber et al (Weber M, JJ Davies, D Wittig, EJ Oakeley, M Haase, WL Lam and D Schubeler. 2005. Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nat Genet 37: 853-862.). Immunoprecipitated DNAs (MeDIP) were purified using silica columns (Zymo Research) and eluted in TE buffer. Forty ng of both Input and MeDIP samples were whole genome amplified (WGA) using the WGA2 kit (Sigma Aldrich) following the manufacturer’s instruction, but omitting the fragmentation step. WGA reactions were cleaned up using silica columns (Sigma Aldrich) and eluted in water. Labeling and hybridization of arrays was performed according to the manufactures’ protocol. In brief, 1 µg of Input DNA and 1 µg of MeDIP DNA were labeled with Cy3 and Cy5 respectively by random priming using the Dual Color DNA labeling kit (Roche NimbleGen). The labeled samples were precipitated using isopropanol and quantified spectrophotometrically using the NanoDrop 1000.
Hybridization protocol
The pellet was dissolved in hybridization solution using the NimbleGen Hybridization kit. After denaturing the probe was hybridized overnight on the 2.1M Deluxe Promoter Arrays using the HX1 mixers and the NimbleGen Hybridization system 4.
Scan protocol
Slides were scanned using the 2 µm high resolution NimbleGen MS 200 micro array scanner per manufacturer's protocol.
Description
Sample name: 471226
Data processing
Signal intensity data was extracted from the scanned images of each array using NimbleScan v2.6 software and quantile normalized on a per channel basis. Log2 ratios of the intensities were computed (ratio of MeDIP signal/Input signal) for each array. Detection of differential methylation was performed using the Probe Sliding Window-ANOVA algorithm (PSW-ANOVA). PSW-ANOVA (Roche NimbleGen) leverages the “neighbor effect” characteristics of Methylation Dependent ImmunoPrecipitation on chip (MeDIP-chip) data from high density oligonucleotide arrays, where enriched fragments sizes far exceed the spacing between probes. In other words, probes within close proximity have a greater likelihood of reporting signals derived from the same enriched fragment during MeDIP procedure. PSW-ANOVA uses a sliding window approach to identify potential sites of differential enrichment and then assigns probability scores (p values) to each probe on the array using a repeated measure ANOVA model. PSW-ANOVA is implemented in the R statistical programming environment as a custom script and was provided by Roche NimbleGen in collaboration. Briefly, for each probe, a sliding window is determined based on the sliding window size parameter, centering on the probe in question. Within each sliding window, a repeated measures ANOVA is used to assess the difference between the various experimental groups for all probes located within the sliding window, using the probe ID as the reference to reduce the amount of error arising from different probes within a sliding window. It is assumed that the background and non-specific hybridization behavior of adjacent probes within a sliding window can be larger than the changes in intensity difference between the two channels, but within the sliding window, the log ratios will still be proportional to the intensity difference between groups of samples. PSW-ANOVA (sliding window of 750 bp comprising 7 probes, and a FDR adjusted p- value < 0.05) was used to identify differential methylated regions (DMR) which were statistically significantly different between the different conditions tested in the experiment. Peaks were identified in the DMR by searching for the number of probes (default = 2) above a p-value minimum cutoff (-log10, default = 1.3) within 150 bp distance of each other. Peaks within 500 bp were merged. Peaks were mapped to promoter regions (from 3 kb upstream to 1 kb downstream of the transcription start site) and CpG islands of genes using the NimbleScan v2.6 software.
