title: Measuring Sister Chromatid Cohesion Protein Genome Occupancy in Drosophila melanogaster by ChIP-seq.
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.
authors: Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, 1100 South Grand Boulevard, Saint Louis, MO, 63104, USA. dorsettd@slu.edu.
authors: Dale
authors: D
authors: Dorsett
authors: Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, 1100 South Grand Boulevard, Saint Louis, MO, 63104, USA.
authors: Ziva
authors: Z
authors: Misulovin
mesh_terms: D000818:Animals; D002842:Chromatids / Q000235:genetics; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D020090:Chromosome Segregation / Q000235:genetics*; D029721:Drosophila Proteins / Q000235:genetics / Q000302:isolation & purification*; D004331:Drosophila melanogaster / Q000235:genetics; D049750:Genome, Insect / Q000235:genetics; D023281:Genomics / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D012854:Sister Chromatid Exchange / Q000235:genetics; D000097722:Cohesins
chemical_list: D029721:Drosophila Proteins; C506513:cohesin protein complex, Drosophila; D000097722:Cohesins
keywords: ChIP-chip; ChIP-seq; Chromatin immunoprecipitation; Cohesin; Drosophila; High-throughput sequencing; Ion Torrent proton; Microarray; Nipped-B