Tuesday, June 8, 2010

Library preparation for ChIP-Seq

While Epicentre’s novel Nextera™ technology is revolutionizing next-generation sequencing library preparation, many laboratories are still using older methods of creating genomic DNA libraries for next-generation sequencing. A recent study (Cheung et al.*) of transcriptional regulation mediated by trimethylated histone H3K4 used ChIP-Seq analysis in samples obtained from the human prefrontal cortex.

Preparation of the ChIP-Seq libraries involved several Epicentre products: the End-It™ DNA End Repair Kit, Exo-Minus Klenow, and the Fast-Link™ DNA Ligation Kit, to end-repair, A-tail, and ligate Illumina Genomic Adaptors to sheared DNA. The end-tagged DNA was PCR-amplified, and sequenced using an Illumina Genome Analyzer II. The researchers report a solid smear of DNA at the 160-230 bp size range, and a secondary smear of less intensity (around 400 bp), which they attributed to dinucleosomal DNA. The sequencing data were used to analyze the number and frequency of epigenomic changes in the prefrontal cortex neurons, with important implications for a variety of neurodevelopmental disorders.

ResearchBlogging.orgCheung, I. et al. (2010). Developmental regulation and individual differences of neuronal H3K4me3 epigenomes in the prefrontal cortex Proceedings of the National Academy of Sciences, 107 (19), 8824-8829 DOI: 10.1073/pnas.1001702107

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