Chapter title |
Measuring Sister Chromatid Cohesion Protein Genome Occupancy in Drosophila melanogaster by ChIP-seq.
|
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Chapter number | 8 |
Book title |
Cohesin and Condensin
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Published in |
Methods in molecular biology, January 2017
|
DOI | 10.1007/978-1-4939-6545-8_8 |
Pubmed ID | |
Book ISBNs |
978-1-4939-6543-4, 978-1-4939-6545-8
|
Authors |
Dale Dorsett, Ziva Misulovin |
Editors |
Kyoko Yokomori, Katsuhiko Shirahige |
Abstract |
This chapter presents methods to conduct and analyze genome-wide chromatin immunoprecipitation of the cohesin complex and the Nipped-B cohesin loading factor in Drosophila cells using high-throughput DNA sequencing (ChIP-seq). Procedures for isolation of chromatin, immunoprecipitation, and construction of sequencing libraries for the Ion Torrent Proton high throughput sequencer are detailed, and computational methods to calculate occupancy as input-normalized fold-enrichment are described. The results obtained by ChIP-seq are compared to those obtained by ChIP-chip (genomic ChIP using tiling microarrays), and the effects of sequencing depth on the accuracy are analyzed. ChIP-seq provides similar sensitivity and reproducibility as ChIP-chip, and identifies the same broad regions of occupancy. The locations of enrichment peaks, however, can differ between ChIP-chip and ChIP-seq, and low sequencing depth can splinter broad regions of occupancy into distinct peaks. |
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