The methylation analysis was performed using EmbryoGENE DNA methylation array (EDMA) platform. The methodology have been thoroughly describef in (Saadi et al., 2014). Briefly, 10ng of DNA sample and spike-in control were cut in ≈160bp fragment using MSEI restriction enzyme. MseLig 12 and MseLig 21 adapters were added to the fragmented gDNA sites. Sample were the digested using methyl sensitive restriction enzyme HpaII, HinP1I and AciI. Only unmethylated fragment have been targeted by those enzyme, leaving only one adapter on the cleaved fragment. Efficiency of the digestion was confirmed by analyzing the spike-in control added prior to the DNA fragmentation step. For the step, qPCR was performed by preparing a separate mixes containing forward and reverse primer targeting HpaII, HinP1I and AciI site. Undigested spike-in (1/1000 dilution) was used as a positive control and non-template controls were also added as negatives control. The amplification plot and the dissociation curve were evaluated for each primer set separately. The DNA fragmentation was considered successful when the threshold cycle (ct) was greater than 5 when compared to the undigested control.Fragment selection by ligation-mediated PCR After cleavage by methyl sensitive enzyme, the gDNA was purified by ethanol precipitation and dissolve in nuclease free water. Selective amplification of methylated fragments was performed using two rounds of ligation-mediated PCR (LM-PCR). The PCR products were resolved on 0.8% agarose gel to assess the quality. Then the previously added linker were removed.
Extracted molecule
genomic DNA
Extraction protocol
One unit of extended semen was diluted with 1ml of phosphate buffered saline, pH 7.4, 2% SDS in a 1.5 ml microcentrifuge tube. The mixture was vortexed 5 seconds and centrifuged at 1550g for 5 minutes to pellet the sperms. Following centrifugation the supernatant was aspirated. 450µl of lysis buffer (10mM Tris pH 8.0, 10mM EDTA pH 8.0, 1.5% SDS, 100mM NaCl) followed by the addition of 30µl of 1M dithiothreitol and 20µl of Proteinase K solution (20mg/ml). The solution was incubated for 12-16 hours at 56°C in heater shaker with the speed of 155-157 rpm with horizontal tubes tighten to the plate by tape. Following incubation, 160 µl of saturated NaCl solution was added. The mixture was then vortexed for 3x 5 seconds. The solution was then centrifuged for 10 minutes at 15500g. The supernatant from the liquid pellet was carefully removed and poured in 2ml microtubes. 1ml of chilled (4°C) ethanol was added and mixed gently by hand (20 times) to allow DNA to precipitate and centrifuged 15 minutes ate 16000g at 4°C. The supernatant was removed and discarded. 500µl of chilled (4°C) ethanol 70% was added and the mixture was centrifuged for 5 minutes at 16000g. The supernatant was removed and discarded. 200µl of chilled (4°C) ethanol 50% was added and the mixture was centrifuged for 5 minutes at 16000g. The purified DNA was suspended in 100µl of TE buffer (10mM Tris-HCl, 1mM EDTA, pH 8.0). Quantity and purity were evaluated using a Nanodrop and validated by migration on 0.5% agarose gel at 75 volt for 1 hour.
Label
cy3
Label protocol
For each sample, 2 μg of DNA was labeled using the Universal Linkage System (ULS) labeling kit (Kreatech Biotechnology) according to the manufacturer’s instructions with minor modifications: 1 μL of Cy-ULS dye was added per 1 μg of genomic DNA adjusted with 10× labeling buffer. The labelling mixture was then held for 30 min at 85°C in a thermocycler, followed by 3 min on ice. Non-reacted ULS-Cy3/5 was removed by purification using a QIAquick PCR Purification Kit and samples were eluted in 23.5 μL nuclease-free water. A 1.5 μL aliquot was used to determine DNA concentration and dye incorporation using the ND- 1000 NanoDrop.
The methylation analysis was performed using EmbryoGENE DNA methylation array (EDMA) platform. The methodology have been thoroughly describef in (Saadi et al., 2014). Briefly, 10ng of DNA sample and spike-in control were cut in ≈160bp fragment using MSEI restriction enzyme. MseLig 12 and MseLig 21 adapters were added to the fragmented gDNA sites. Sample were the digested using methyl sensitive restriction enzyme HpaII, HinP1I and AciI. Only unmethylated fragment have been targeted by those enzyme, leaving only one adapter on the cleaved fragment. Efficiency of the digestion was confirmed by analyzing the spike-in control added prior to the DNA fragmentation step. For the step, qPCR was performed by preparing a separate mixes containing forward and reverse primer targeting HpaII, HinP1I and AciI site. Undigested spike-in (1/1000 dilution) was used as a positive control and non-template controls were also added as negatives control. The amplification plot and the dissociation curve were evaluated for each primer set separately. The DNA fragmentation was considered successful when the threshold cycle (ct) was greater than 5 when compared to the undigested control.Fragment selection by ligation-mediated PCR After cleavage by methyl sensitive enzyme, the gDNA was purified by ethanol precipitation and dissolve in nuclease free water. Selective amplification of methylated fragments was performed using two rounds of ligation-mediated PCR (LM-PCR). The PCR products were resolved on 0.8% agarose gel to assess the quality. Then the previously added linker were removed.
Extracted molecule
genomic DNA
Extraction protocol
One unit of extended semen was diluted with 1ml of phosphate buffered saline, pH 7.4, 2% SDS in a 1.5 ml microcentrifuge tube. The mixture was vortexed 5 seconds and centrifuged at 1550g for 5 minutes to pellet the sperms. Following centrifugation the supernatant was aspirated. 450µl of lysis buffer (10mM Tris pH 8.0, 10mM EDTA pH 8.0, 1.5% SDS, 100mM NaCl) followed by the addition of 30µl of 1M dithiothreitol and 20µl of Proteinase K solution (20mg/ml). The solution was incubated for 12-16 hours at 56°C in heater shaker with the speed of 155-157 rpm with horizontal tubes tighten to the plate by tape. Following incubation, 160 µl of saturated NaCl solution was added. The mixture was then vortexed for 3x 5 seconds. The solution was then centrifuged for 10 minutes at 15500g. The supernatant from the liquid pellet was carefully removed and poured in 2ml microtubes. 1ml of chilled (4°C) ethanol was added and mixed gently by hand (20 times) to allow DNA to precipitate and centrifuged 15 minutes ate 16000g at 4°C. The supernatant was removed and discarded. 500µl of chilled (4°C) ethanol 70% was added and the mixture was centrifuged for 5 minutes at 16000g. The supernatant was removed and discarded. 200µl of chilled (4°C) ethanol 50% was added and the mixture was centrifuged for 5 minutes at 16000g. The purified DNA was suspended in 100µl of TE buffer (10mM Tris-HCl, 1mM EDTA, pH 8.0). Quantity and purity were evaluated using a Nanodrop and validated by migration on 0.5% agarose gel at 75 volt for 1 hour.
Label
Cy5
Label protocol
For each sample, 2 μg of DNA was labeled using the Universal Linkage System (ULS) labeling kit (Kreatech Biotechnology) according to the manufacturer’s instructions with minor modifications: 1 μL of Cy-ULS dye was added per 1 μg of genomic DNA adjusted with 10× labeling buffer. The labelling mixture was then held for 30 min at 85°C in a thermocycler, followed by 3 min on ice. Non-reacted ULS-Cy3/5 was removed by purification using a QIAquick PCR Purification Kit and samples were eluted in 23.5 μL nuclease-free water. A 1.5 μL aliquot was used to determine DNA concentration and dye incorporation using the ND- 1000 NanoDrop.
Hybridization protocol
Hybridizations were performed according to the microarray manufacturer’s instructions (Agilent Technologies). Briefly, 1.4 μg of labeled sample (in 40 μL) was mixed with 158 μL of hybridization master mix containing 25 μL of Bovine Hybloc competitor DNA (1 mg/ml) , Applied Genetics Laboratories), 2.6 μL of Agilent 100x Blocking Agent and 130 μL of Agilent 2× HI-RPM Hybridization Buffer (Agilent Technologies). Samples were held at 95°C for 3 min and at 37°C for 30 min followed by addition of 65 μL of Agilent-CGHBlock (final volume 260 μL). The samples were loaded onto the microarray and hybridization was carried out in a hybridization oven (Shel Lab) for 40 h at 65°C and 20 rpm. Washing was carried out according to the microarray manufacturer’s instructions
Scan protocol
Slides were scanned with the PowerScanner (Tecan) and analyzed with Array-Pro Analyzer 6.3 software (MediaCybernetics).
Description
Biological replicats 2 of 4
Data processing
The array data analysis methods and downstream analysis pipelines are described in detail in (Saadi et al., 2014). Briefly, after Loess normalization, quantile inter-array scale normalization was performed and fitted to a linear model and thus, Bayesian statistics of differential expression were obtained. Thus, differentially methylated probes were identified using linear models for microarray data (LIMMA).
