RESUMEN

High-throughput targeted repeat element bisulfite sequencing (HT-TREBS) is designed to assay the methylation level of individual retrotransposon loci of a targeted family, in a locus-specific manner, and on a genome-wide scale. Briefly, genomic DNA is sheared and ligated to Ion Torrent A adaptors (with methylated cytosines), followed by bisulfite-conversion, and amplification with primers designed to bind the targeted retrotransposon. Since the primers carry the Ion Torrent P1 adaptor as a 5'-extension, the amplified library is ready to be size-selected and sequenced on a next-generation sequencing platform. Once sequenced, each retrotransposon is mapped to a particular genomic locus, which is achieved through ensuring at least a 10-bp overlap with flanking unique sequence, followed by the calculation of methylation levels of the mapped retrotransposon using a BiQ Analyzer HT. A complete protocol for library construction as well as the bioinformatics for HT-TREBS is described in this chapter.

Asunto(s)

RESUMEN

DNA methylation is a major epigenetic mark associated with multiple aspects of retrotransposons within the mammalian genome. In order to study DNA methylation of a large number of retrotransposons on an individual-locus basis, we have developed a new protocol termed High-Throughput Targeted Repeat Element Bisulfite Sequencing (HT-TREBS) (Ekram and Kim, 2014 [1]). We have used this technique to characterize the locus-specific patterns of DNA methylation of 4799 members of the mouse IAP LTR (Intracisternal A Particle Long Terminal Repeat) retrotransposon family in embryonic stem, somatic and Neuro2A cells (Bakshi and Kim, 2014 [2]). Here we describe in detail the sample preparation and bioinformatics analyses used for these studies. The somatic cell data may be accessed under GEO accession number GSE49222. The ES and Neuro2A data are deposited under GEO accession number GSE60007.