|
| Status |
Public on May 10, 2016 |
| Title |
PHH_WO_control_exp2 [genomic] |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Input DNA from PHH exposed to zero dose of AFB1 for 5 days followed by a 3-day washout period replicate 2
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: primary human hepatocytes
fraction: input
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were lysed in 500 µl digestion buffer (containing 0.5 M EDTA; 1 M Tris–HCl, pH 8.0; 10% SDS) and incubated for 1 hour at 55 °C. Next, 25µl of proteinase K (20mg/ml) (Ambion) was added. After incubation of 1 hour at 55 °C, the proteinase K was inactivated for 10 minutes at 80 °C. RNAse A (2 µl; 100mg/ml) (Qiagen) and collagenase (25 µl; 1%) (Sigma) treatment was performed for 1 hour at 37 °C. Thereupon, 500 µl of phenol-chloroform-isoamylalcohol (PCI; 25:24:1) (Sigma) was added. The mixture was shaked manually for 5 min, and centrifuged for 5 minutes at maximum speed. The upper phase was transferred to a new Eppendorf and the step with PCI was repeated. The upper phase was collected and precipitated using 50 µl 3M NaAc pH 5.6 and 1250 µl cold 100% ethanol for 30 min. at -80°C. After centrifugation for 30 minutes at maximum speed, the DNA pellet was washed using cold 70% ETOH, dried in a speed vac and dissolved in 50µl nuclease free water. The total amount was at least 10 µg DNA, the 260/280 ratio ranged between 1.7-1.9, and the 260/230 ratio appeared higher than 1.6. A total of 6 DNA samples were prepared.
|
| Label |
Cy3
|
| Label protocol |
In order to obtain fragments ranging from 200 bp to 600 bp, genomic DNA was sonicated. Next, the fragments were cleaned up using silica columns (Zymo Research) and eluted in TE buffer. MeDIP was performed using the MagMeDIP kit (Diagenode, Liege, Belgium) according to the manufacturer’s protocol. Briefly, IP incubation mix was added to 1.2 µg sonicated DNA sample and denaturated at 95°C. 10% of this was used as reference samples. The remaining sample was immunoprecipitated overnight using antibody mix containing the 5-methylcytidine antibody and magnetic beads. Following purification using the Ipure kit (Diagenode), reference and MeDIP samples were prepared for microarray analysis by whole genome amplification (WGA) using the WGA2 kit (Sigma-Aldrich) without performing the fragmentation step. Methylation enrichment in the paired samples MeDIP/reference was derived from qPCR data by calculating the ratio positive control/negative control, applying the ΔΔCt method. The Human DNA Methylation 2.1M Deluxe Promoter Array (Roche NimbleGen) was used for analyses of DNA methylation levels.
|
| |
|
| Channel 2 |
| Source name |
DNA from PHH exposed to zero dose of AFB1 for 5 days followed by a 3-day washout period replicate 2
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: primary human hepatocytes
fraction: Immunoprecipitated genomic DNA with antibody against a monoclonal antibody against 5’-methylcytidine
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were lysed in 500 µl digestion buffer (containing 0.5 M EDTA; 1 M Tris–HCl, pH 8.0; 10% SDS) and incubated for 1 hour at 55 °C. Next, 25µl of proteinase K (20mg/ml) (Ambion) was added. After incubation of 1 hour at 55 °C, the proteinase K was inactivated for 10 minutes at 80 °C. RNAse A (2 µl; 100mg/ml) (Qiagen) and collagenase (25 µl; 1%) (Sigma) treatment was performed for 1 hour at 37 °C. Thereupon, 500 µl of phenol-chloroform-isoamylalcohol (PCI; 25:24:1) (Sigma) was added. The mixture was shaked manually for 5 min, and centrifuged for 5 minutes at maximum speed. The upper phase was transferred to a new Eppendorf and the step with PCI was repeated. The upper phase was collected and precipitated using 50 µl 3M NaAc pH 5.6 and 1250 µl cold 100% ethanol for 30 min. at -80°C. After centrifugation for 30 minutes at maximum speed, the DNA pellet was washed using cold 70% ETOH, dried in a speed vac and dissolved in 50µl nuclease free water. The total amount was at least 10 µg DNA, the 260/280 ratio ranged between 1.7-1.9, and the 260/230 ratio appeared higher than 1.6. A total of 6 DNA samples were prepared.
|
| Label |
Cy5
|
| Label protocol |
In order to obtain fragments ranging from 200 bp to 600 bp, genomic DNA was sonicated. Next, the fragments were cleaned up using silica columns (Zymo Research) and eluted in TE buffer. MeDIP was performed using the MagMeDIP kit (Diagenode, Liege, Belgium) according to the manufacturer’s protocol. Briefly, IP incubation mix was added to 1.2 µg sonicated DNA sample and denaturated at 95°C. 10% of this was used as reference samples. The remaining sample was immunoprecipitated overnight using antibody mix containing the 5-methylcytidine antibody and magnetic beads. Following purification using the Ipure kit (Diagenode), reference and MeDIP samples were prepared for microarray analysis by whole genome amplification (WGA) using the WGA2 kit (Sigma-Aldrich) without performing the fragmentation step. Methylation enrichment in the paired samples MeDIP/reference was derived from qPCR data by calculating the ratio positive control/negative control, applying the ΔΔCt method. The Human DNA Methylation 2.1M Deluxe Promoter Array (Roche NimbleGen) was used for analyses of DNA methylation levels.
|
| |
|
| |
| Hybridization protocol |
Labeling and hybridization of arrays were performed according to the manufactures’ protocol. Slides were washed using the NimbleGen wash buffer kit and scanned using the 2 µm high resolution NimbleGen MS 200 microarray scanner.
|
| Scan protocol |
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.
|
| Description |
563247
|
| Data processing |
Log2 ratios of the intensities were computed (ratio of MeDIP signal / Input signal) and for each array, centering was performed by subtracting the global array bi-weight mean of the log2 ratios such that the computed log2 ratios were centered around 0. Detection of differential methylation was performed using the Probe Sliding Window-ANOVA algorithm (PSW-ANOVA). PSW-ANOVA was implemented in the R statistical programming environment (v2.15.3) (http://www.r-project.org) as a custom script and was provided by Roche NimbleGen in collaboration . PSW-ANOVA (sliding window of 750 bp comprising 7 probes, and a FDR corrected p- value < 0.01) was used to identify differential methylated regions (DMR) which were statistically significantly different between the different conditions tested in the experiment i.e. Exposed versus Control. Peaks were identified in the DMR by searching for regions containing at least 8 significant consecutive probes (p<0.01). Peaks were mapped to promoter regions (from 3 kb upstream to 1 kb downstream of the transcription start site (25)) and CpG islands of genes using the NimbleScan v2.6 software. A control corrected median log2 ratio was calculated for each peak. Log2 ratio’s > 0 indicate hypermethylation and log2 ratio’s < 0 indicate hypomethylation.
scaled, log2 (medip/Input) ratio for demethylated regions
|
| |
|
| Submission date |
Jul 30, 2015 |
| Last update date |
May 10, 2016 |
| Contact name |
Linda Rieswijk |
| E-mail(s) |
linda.rieswijk@maastrichtuniversity.nl
|
| Organization name |
Maastricht University
|
| Department |
Toxicogenomics
|
| Street address |
Universiteitssingel 50
|
| City |
Maastricht |
| ZIP/Postal code |
6229 ER |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL16284 |
| Series (2) |
| GSE71548 |
Aflatoxin B1 induces persistent epigenomic effects in primary human hepatocytes associated with hepatocellular carcinoma [DNA methylation] |
| GSE71549 |
Aflatoxin B1 induces persistent epigenomic effects in primary human hepatocytes associated with hepatocellular carcinoma |
|
