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| Status |
Public on Dec 15, 2017 |
| Title |
Primary human hepatocyte 8 days control replicate 1 |
| Sample type |
genomic |
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| Channel 1 |
| Source name |
input DNA from human primary hepatocytes
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Human primary hepatocytes
treatment: exposed to 1% ethalnol for 5 days daily and subsequently to 3 days medium
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were lysed in 500 µl of 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, 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 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 of 3M NaAc pH 5.6 and 1250 µl of 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 of 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 12 DNA samples were prepared. DNA was used for MeDIP-Chip analyses.
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| Label |
Cy3
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| Label protocol |
Genomic DNA was sonicated to obtain fragments ranging from 200 bp to 600 bp, cleaned up using silica columns (Zymo Research) and eluted in TE buffer. MeDIP was performed using the MagMeDIP kit (Diagenode, Liège, Belgium) according to the manufacturer’s protocol. In brief, IP incubation mix (containing magbuffer A, magbuffer B, 1.5 µg methylated DNA positive control, 1.5 µg non-methylated DNA negative control) was added to 1.2 µg of sonicated sample and denatured at 95 °C. 10% of this was kept aside as Input sample and stored at 4°C. Beads were washed 3 times with ice-cold diluted magbuffer A and collected in 20 µl of diluted magbuffer A. The remaining sample was immunoprecipitated using 5µl of antibodymix (containing an antibody against 5’-methylcytidine (provided in the kit) magbuffer A and magbuffer C) and 20 µl of magnetic beads solution. Immunoprecipitation was performed overnight at 4 °C on a rotating wheel. The following day, magnetic beads were washed twice with wash buffer 1 and once with wash buffer 2 and kept on ice. Next, DNA isolation was performed using the IPure kit (Diagenode, Liège, Belgium). Fifty ul of elution buffer was added to the MeDIP-bead pellet and 92.5 µl to the Input sample and incubated for 15 min at room temperature on a rotating wheel. Supernatant was transferred to a new Eppendorf tube using a magnet. Samples were subsequently incubated with 2 µl of glycogen carrier, 100 µl of 100% isopropanol and 15 µl of magnetic beads and incubated for 1 hour at room temperature on a rotating wheel. Magnetic beads were washed using wash buffer 1 and wash buffer 2 for 5 minutes. The DNA was eluted twice by using 75 µl of buffer C and incubated for 15 min at room temperature on a rotating wheel. All the samples were precipitated with sodium acetate and ethanol with glycogen as a carrier for 30 min at -80 °C and centrifuged at max speed for 30 min at 4°C. The cell pellet was washed using 70% ethanol and after air drying suspended in 20µl MQ. Eight µl of both Input and MeDIP samples were used for amplification by whole genome amplification (WGA) using the WGA2 kit (Sigma Aldrich) following the manufacturer’s instruction, without performing the fragmentation step. WGA reactions were cleaned up using silica columns (Sigma Aldrich) and eluted in water. Methylation enrichment in the paired samples MeDIP/Input was derived from qPCR data by calculating the ratio positive control/negative control, applying the ΔΔCq method using the primers included in the kit.
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| Channel 2 |
| Source name |
medip DNA from human primary hepatocytes
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Human primary hepatocytes
treatment: exposed to 1% ethalnol for 5 days daily and subsequently to 3 days medium
antibody: antibody against 5'-methylcytidine
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were lysed in 500 µl of 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, 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 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 of 3M NaAc pH 5.6 and 1250 µl of 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 of 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 12 DNA samples were prepared. DNA was used for MeDIP-Chip analyses.
|
| Label |
Cy5
|
| Label protocol |
Genomic DNA was sonicated to obtain fragments ranging from 200 bp to 600 bp, cleaned up using silica columns (Zymo Research) and eluted in TE buffer. MeDIP was performed using the MagMeDIP kit (Diagenode, Liège, Belgium) according to the manufacturer’s protocol. In brief, IP incubation mix (containing magbuffer A, magbuffer B, 1.5 µg methylated DNA positive control, 1.5 µg non-methylated DNA negative control) was added to 1.2 µg of sonicated sample and denatured at 95 °C. 10% of this was kept aside as Input sample and stored at 4°C. Beads were washed 3 times with ice-cold diluted magbuffer A and collected in 20 µl of diluted magbuffer A. The remaining sample was immunoprecipitated using 5µl of antibodymix (containing an antibody against 5’-methylcytidine (provided in the kit) magbuffer A and magbuffer C) and 20 µl of magnetic beads solution. Immunoprecipitation was performed overnight at 4 °C on a rotating wheel. The following day, magnetic beads were washed twice with wash buffer 1 and once with wash buffer 2 and kept on ice. Next, DNA isolation was performed using the IPure kit (Diagenode, Liège, Belgium). Fifty ul of elution buffer was added to the MeDIP-bead pellet and 92.5 µl to the Input sample and incubated for 15 min at room temperature on a rotating wheel. Supernatant was transferred to a new Eppendorf tube using a magnet. Samples were subsequently incubated with 2 µl of glycogen carrier, 100 µl of 100% isopropanol and 15 µl of magnetic beads and incubated for 1 hour at room temperature on a rotating wheel. Magnetic beads were washed using wash buffer 1 and wash buffer 2 for 5 minutes. The DNA was eluted twice by using 75 µl of buffer C and incubated for 15 min at room temperature on a rotating wheel. All the samples were precipitated with sodium acetate and ethanol with glycogen as a carrier for 30 min at -80 °C and centrifuged at max speed for 30 min at 4°C. The cell pellet was washed using 70% ethanol and after air drying suspended in 20µl MQ. Eight µl of both Input and MeDIP samples were used for amplification by whole genome amplification (WGA) using the WGA2 kit (Sigma Aldrich) following the manufacturer’s instruction, without performing the fragmentation step. WGA reactions were cleaned up using silica columns (Sigma Aldrich) and eluted in water. Methylation enrichment in the paired samples MeDIP/Input was derived from qPCR data by calculating the ratio positive control/negative control, applying the ΔΔCq method using the primers included in the kit.
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| Hybridization protocol |
For analysis of DNA methylation levels, the Human DNA Methylation 2.1M Deluxe Promoter Array (Roche NimbleGen) was used. This array has a density of 2.1 million probes (50-75 oligonucleotides long, median probe spacing 100 bp) that represent all annotated human promoters (~ 26,210), 27,867 CpG islands, and 750 miRNA promoters per slide. The promoters and CpG islands largely overlap with transcription start sites of well characterized RefSeq genes, covering, on average, 8 kb upstream and 3 kb downstream. 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. Thirty-four microgram of Cy3 Input and 34 µg of Cy5 MeDIP DNA were pooled and completely dried by using a speed vac. 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. Slides were washed using the NimbleGen wash buffer kit and scanned using the 2 µm high resolution NimbleGen MS 200 micro array scanner.
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| Scan protocol |
Slides were scanned using the 2 µm high resolution NimbleGen MS 200 micro array scanner per manufacturer's protocol.
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| Description |
545029
PHH_exposed_to_VPA_for_5_days_daily_including_3_days_washout.txt
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| 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) 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. 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 (20) 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 hyper methylation and log2 ratio’s < 0 indicate hypo methylation.
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| Submission date |
Jul 08, 2016 |
| Last update date |
Dec 15, 2017 |
| Contact name |
Simone G van Breda |
| E-mail(s) |
s.vanbreda@maastrichtuniversity.nl
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| Phone |
0031433882127
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| Organization name |
Maastricht University
|
| Department |
Toxicogenomics
|
| Street address |
Universiteitssingel 50
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| City |
Maastricht |
| State/province |
Limburg |
| ZIP/Postal code |
6229 ER |
| Country |
Netherlands |
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| Platform ID |
GPL16284 |
| Series (2) |
| GSE84189 |
Induction of DNA methylation changes in primary human hepatocytes by valproic acid |
| GSE84250 |
Induction of changes in primary human hepatocytes by valproic acid |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM2228810_545029_532.pair.gz |
42.7 Mb |
(ftp)(http) |
PAIR |
| GSM2228810_545029_635.pair.gz |
42.3 Mb |
(ftp)(http) |
PAIR |
| Processed data are available on Series record |
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