RNAi depletion of Chromatin proteins (S2-DRSC cells)

Depletion of Non-histone Chromatin proteins with RNAi (Karpen project, Elgin subgroup)

Nuclei were isolated from the Drosophila S2 embryonic cell line that had been transfected with dsRNA targeting the jellyfish green fluorescent protein (GFP) gene, and crosslinked with formaldehyde. After lysis and sonication the chromatin was immunoprecipitated with an affinity-purified antibody that recognizes chromosomal protein HP1. 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).

General Description

The goal of these experiments are to validate and confirm the locations of 125 chromosomal proteins across the Drosophila melanogaster genome. To do this, we are using RNAi to deplete individual non-histone chromosomal proteins in Drosophila BG3 and S2 tissue culture cells, and then using antibodies to perform Chromatin ImmunoPrecipitation (ChIP) using 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: Hybridization to Affy arrays, WGA, ChIP, Array Scanning Protocol
  3. Data Analysis: M-value normalization, Smoothed M-value enrichment profiles
  4. Other Protocols: RNAi treatment in cell culture, Regions of significant enrichment

Release Date: 2011-08-30