RNAi Depletion of Histone Variants (S2-DRSC cells)

H2AV_9751.S2.H2AV_RNAi.Solexa (Karpen project, Karpen subgroup)

Details

Nuclei were isolated from the Drosophila S2 embryonic cell line that had been transfected with dsRNA targeting the His2Av gene, and crosslinked with formaldehyde. After lysis and sonication the chromatin was immunoprecipitated with an affinity-purified antibody that recognizes histone H2A variant. The resulting fragments were used to create libraries and sequenced on the Illumina platform. Short reads were aligned to the reference genome using Bowtie, and peaks for each replicate were called using SPP (using sequence from the Input material as a comparison control). Statistically significant regions of enrichment from the two biological replicates were then combined and used to call the cumulative peaks.

General Description

The goal of these experiments are to a) validate/confirm the locations of 125 chromosomal proteins across the Drosophila melanogaster genome and b) evaluate their biological significance by assaying the impact of depletion on other proteins/marks. We are using RNAi to deplete individual non-histone chromosomal proteins in Drosophila BG3 and S2 tissue culture cells, followed by Chromatin ImmunoPrecipitation (ChIP) assayed on genomic tiling arrays. Comparison of a protein factor's binding profiles before and after depletion will increase the confidence of our predictions.

Protocols

  1. Growth and isolation: RNAi treatment in cell culture, Chromatin prep from cultured cells
  2. Sample preparation: Illumina sequencing, ChIP
  3. Data Analysis: Bowtie alignment
  4. Other Protocols: SPP Peak calling

Experimental Reagents

    Antibodies:

Sample Details

    Animals/Lines:


Release Date: 2013-03-25 Submission 5595 Release Date: 2013-03-25 Submission 5595