In mammals, DNA methylation is essential to life. Methylation occurs largely (but not exclusively) in the context of a simple palindromic dinucleotide sequence, 5'CG 3'. Important biological properties of DNA methylation are currently poorly understood, including its relationship to gene function. We have chosen to map DNA methylation patterns across the genome in an adult tissue from inbred strains of mice. Our goals are to understand the relationship(s) between methylation patterns and DNA sequence as well as genomic features. We are using whole genome bisulfite sequencing to determine methylation patterns in our biological system of choice. We couple this data with RNA Sequence data from the same tissue samples to ascertain the relationship, if any, between DNA methylation and gene expression. The experimental design includes examination of methylation in liver tissue using inbred mice. We have chosen to cross C57B6 mice with C3H mice. We mated B6 males with C3 females (and also performed the reciprocal cross). Parents and offspring were sacrificed at identical ages and tissues were collected. In the current funding period, we have isolated high quality total RNA from each animal in the study (24 animals total) and performed RNA-seq. In addition, we prepared high quality genomic DNA and resequenced the genome of representative animals from our study. Finally, we have established a robust method for shotgun genomic bisulfite sequencing. These studies have optimized the molecular biology methods and established analysis platforms to permit deep sequence coverage of the methylome in these animals. We anticipate rapid progress on this project in the coming year.

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