To minimize genetic noise the inbred C57BL/6 strain was used for generation of experimental males and for analysis of the sperm epigenome. Mice were housed under a controlled light/dark cycle and were provided with food and water ad libitum. All animal procedures were approved by the Animal Care and Use Committee of McGill University, Montreal. Dietary exposures began in utero. To generate experimental males, female C57BL/6 were fed either the folate sufficient (FS, 2mg folic acid/kg, n=69) diet (TD.01369, Harlan Laboratories, Madison, WI) or the folate deficient diet (FD, 0.3mg folic acid/kg, n=64) (TD.01546), two weeks prior to breeding with non-experimental C57BL/6 males that were fed regular mouse chow (8640 Rodent diet) (used only for breeding to generate experimental males). To breed C57BL/6 females, C57BL/6 males were brought to the females’ cages at night and removed in the morning in order to limit consumption of the experimental diets by the males. Females were maintained on the experimental diets through pregnancy and lactation. From weaning at PND21, male pups were given the same experimental diet as their in utero exposure until sacrifice as adults.
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA was extracted with a DNeasy Mini Kit (Qiagen, Mississauga, Canada) from mouse sperm.
Label
Cy5
Label protocol
Both immunoprecipitated DNA and input DNA from MeDIP were amplified with the GenomePlex Complete Whole Genome Amplification Kit (Sigma, WGA2) according to the manufacturer’s instructions. Cy3-dUTP and Cy5-dUTP were used by Nimblegen to label the input and bound factions, respectively.
To minimize genetic noise the inbred C57BL/6 strain was used for generation of experimental males and for analysis of the sperm epigenome. Mice were housed under a controlled light/dark cycle and were provided with food and water ad libitum. All animal procedures were approved by the Animal Care and Use Committee of McGill University, Montreal. Dietary exposures began in utero. To generate experimental males, female C57BL/6 were fed either the folate sufficient (FS, 2mg folic acid/kg, n=69) diet (TD.01369, Harlan Laboratories, Madison, WI) or the folate deficient diet (FD, 0.3mg folic acid/kg, n=64) (TD.01546), two weeks prior to breeding with non-experimental C57BL/6 males that were fed regular mouse chow (8640 Rodent diet) (used only for breeding to generate experimental males). To breed C57BL/6 females, C57BL/6 males were brought to the females’ cages at night and removed in the morning in order to limit consumption of the experimental diets by the males. Females were maintained on the experimental diets through pregnancy and lactation. From weaning at PND21, male pups were given the same experimental diet as their in utero exposure until sacrifice as adults.
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA was extracted with a DNeasy Mini Kit (Qiagen, Mississauga, Canada) from mouse sperm.
Label
Cy3
Label protocol
Both immunoprecipitated DNA and input DNA from MeDIP were amplified with the GenomePlex Complete Whole Genome Amplification Kit (Sigma, WGA2) according to the manufacturer’s instructions. Cy3-dUTP and Cy5-dUTP were used by Nimblegen to label the input and bound factions, respectively.
Hybridization protocol
Array hybridization to the NimbleGen mouse 2.1 deluxe promoter array was carried out by Nimblegen..
Scan protocol
Scanning was performed by Nimblegen.
Data processing
The value is background subtracted and log2-quantile normalized using the limma Bioconductor package.