H3 Histone modifications in Oregon strain
H3 Histone modifications in Oregon strain (Karpen Project, Pirrotta Subgroup)
Nuclei were isolated from staged Drosophila Oregon R embryos, 2-4 hr after egg laying that had been crosslinked with formaldehyde. After lysis and sonication the chromatin was immunoprecipitated with an affinity-purified antibody that recognizes histone H3 trimethylated on lysine 4. After PCR amplification and labeling the DNA was used to probe Affymetrix microarrays (a sample of the DNA input for the ChIP was processed in parallel and used to probe another copy of the array). The experiment was performed in triplicate, and normalized, averaged data is shown. For tracks displaying binding peaks, normalized signal intensities from three averaged experimental replicates were analyzed using a peak-calling algorithm that calculates genomic regions of significant enrichment (or depletion).
We aim to determe the locations of 125 chromosomal proteins and histone modifications across the Drosophila melanogaster genome. The proteins and modifications under study are involved in basic chromosomal functions such as DNA replication, gene expression, gene silencing, and inheritance. We will perform Chromatin ImmunoPrecipitation (ChIP) using genomic tiling arrays. We will initially assay localizations using chromatin from three cell lines and two embryonic stages, and will then extend the analysis of a subset of proteins to four additional animal tissues/stages.
- Growth and isolation: Cage population, Cage population, Chromatin prep from fixed D.m. embryos, Chromatin prep from fixed D.m. embryos, Chromatin prep from tissues, Fly head collection
- Sample preparation: Hybridization to Affy arrays, WGA, ChIP, Array Scanning Protocol
- Data Analysis: Regions of significant enrichment, Regions of significant enrichment, Smoothed M-value enrichment profiles, M-value normalization, Smoothed M-value enrichment profiles
- Antibodies: H3K4me2, H3K36me3, H3K9me3 (new lot), H3K4Me3(LP), H3K9me2-Ab2 (new lot), H3K18Ac (new lot)
- Arrays: Affymetrix Drosophila Tiling Arrays v2.0R
- Animals/Lines: Embyro 14-16hr OR, Mixed Adults 22 days AEL, Oregon-R-modENCODE, Embryo 2-4hr OR
- Other Protocols: Regions of significant enrichment