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Sample GSM2230692 Query DataSets for GSM2230692
Status Public on Dec 06, 2016
Title CsA_day3_rep2
Sample type genomic
 
Channel 1
Source name PHH_VPA_day3
Organism Homo sapiens
Characteristics cell type, sample type: primary human hepatocytes, input
treatment: 30µM CsA
time point: day 3
Extracted molecule genomic DNA
Extraction protocol PHH were lysed in 500 µl of digestion buffer (containing 1 mM EDTA; 50 mM Tris–HCl, pH 8.0; 0.5% SDS). Next, 25 µl 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 shaken manually for 5 minutes, 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 of 3M NaAc pH 5.6 and 1250 µl of cold 100% ethanol for 30 min. at -80°C. After centrifugation for 30 min. at maximum speed, the DNA pellet was washed using cold 70% ETOH, dried in a speed-vac and dissolved in 50µl of nuclease free water. The total amount was at least 8 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 18 DNA samples were prepared. DNA was used for MeDIP-Chip analyses.
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 PHH_VPA_day3
Organism Homo sapiens
Characteristics cell type, sample type: primary human hepatocytes, antibody against a monoclonal antibody against 5'-methylcytidine
treatment: 30µM CsA
time point: day 3
Extracted molecule genomic DNA
Extraction protocol PHH were lysed in 500 µl of digestion buffer (containing 1 mM EDTA; 50 mM Tris–HCl, pH 8.0; 0.5% SDS). Next, 25 µl 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 shaken manually for 5 minutes, 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 of 3M NaAc pH 5.6 and 1250 µl of cold 100% ethanol for 30 min. at -80°C. After centrifugation for 30 min. at maximum speed, the DNA pellet was washed using cold 70% ETOH, dried in a speed-vac and dissolved in 50µl of nuclease free water. The total amount was at least 8 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 18 DNA samples were prepared. DNA was used for MeDIP-Chip analyses.
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.
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 as described before [25]. 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 genomic features, e.g. transcription start sites (TSS) region (100bp upstream of TSS to 1000bp downstream of TSS), promoter regions (3kb upstream of TSS to 100bp upstream of TSS) or CpG islands, applying a window from 3 kb upstream to 1 kb downstream by using the NimbleScan v2.6 software [26]. A control corrected median log2 ratio was calculated for each peak. Log2 ratio’s > 0 indicate hyper methylation and log2 ratio’s < 0 indicate hypo methylation
 
Submission date Jul 11, 2016
Last update date Dec 06, 2016
Contact name Jarno Wolters
E-mail(s) j.wolters@maastrichtuniversity.nl
Organization name Maastricht University
Department Toxicogenomics
Street address Universiteitssingel 40
City Maastricht
ZIP/Postal code PO BOX 616/ 6200MD
Country Netherlands
 
Platform ID GPL16284
Series (2)
GSE84276 Integrative "-omics" analysis in primary human hepatocytes unravels persistent mechanisms of cyclosporine A-induced cholestasis (MeDIP)
GSE84281 Integrative "-omics" analysis in primary human hepatocytes unravels persistent mechanisms of cyclosporine A-induced cholestasis

Supplementary file Size Download File type/resource
GSM2230692_563174_Sample_5_DNA-2CSA30uM-3d_532.pair.gz 42.4 Mb (ftp)(http) PAIR
GSM2230692_563174_Sample_5_DNA-2CSA30uM-3d_635.pair.gz 42.3 Mb (ftp)(http) PAIR
Processed data are available on Series record
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