| Autor: |
Bakshi A; Department of Biological Sciences, Louisiana State University, Baton Rouge, LA, USA., Ekram MB; Department of Biological Sciences, Louisiana State University, Baton Rouge, LA, USA., Kim J; Department of Biological Sciences, Louisiana State University, Baton Rouge, LA, USA. jkim@lsu.edu. |
| Jazyk: |
angličtina |
| Zdroj: |
Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2019; Vol. 1908, pp. 219-228. |
| DOI: |
10.1007/978-1-4939-9004-7_15 |
| Abstrakt: |
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. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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