|
| Status |
Public on Apr 02, 2020 |
| Title |
Blastocysts_16_months_rep3 (10 vs. 16 m) |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Blastocysts
|
| Organism |
Bos taurus |
| Characteristics |
sample type: IVF with sperm from a 16 months old bull
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Total RNA and gDNA were extracted using the AllPrep DNA/RNA Micro Kit (Qiagen)
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.
|
| 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.
|
| |
|
| Channel 2 |
| Source name |
Blastocysts
|
| Organism |
Bos taurus |
| Characteristics |
sample type: IVF with sperm from a 10 months old bull
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Total RNA and gDNA were extracted using the AllPrep DNA/RNA Micro Kit (Qiagen)
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.
|
| 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 |
Scan on a PowerScanner (Tecan) at 2um using the autogain function. Images were quantified using ArrayPro version 6.3 (MediaCybernetics)
|
| Description |
Biological replicates 3 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).
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| |
|
| Submission date |
Mar 15, 2019 |
| Last update date |
Apr 03, 2020 |
| Contact name |
Marc-André Sirard |
| E-mail(s) |
[email protected]
|
| Organization name |
Université Laval
|
| Department |
Sciences Animales
|
| Street address |
Offfice 2732, 2440 Hochelaga Blvd.
|
| City |
Québec City |
| State/province |
Quebec |
| ZIP/Postal code |
G1V 0A6 |
| Country |
Canada |
| |
|
| Platform ID |
GPL18384 |
| Series (2) |
| GSE128353 |
DNA methylation analysis of blastocyst according to peripubertal age of bull (10 vs 16 months) |
| GSE128629 |
The age of the bull influences the transcriptome and epigenome of the blastocysts |
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