Chrom. Silencers and Re-modellers in Oregon strain

dRING Q3200.2-4 hr OR embryo.Affy 2 (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 the chromosomal silencing protein Sex combs extra. 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).

Details

General Description

We aim to determine the locations of the major histone modifications
across the Drosophila melanogaster genome. The 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.

Protocols

  1. Growth and isolation: Cage population, Chromatin prep from fixed D.m. embryos
  2. Sample preparation: ChIP, WGA, Hybridization to Affy arrays, Array Scanning Protocol
  3. Data Analysis: M-value normalization, Smoothed M-value enrichment profiles
  4. Other Protocols: Regions of significant enrichment

Experimental Reagents

  1. Antibodies: dRING

Sample Details

  1. Animals/Lines: Drosophila melanogaster


Release Date: 2013-02-28