RNAi depletion of Chromatin proteins (ML-DmBG3-C2 cells)
Depletion of Non-histone Chromatin Proteins with RNAi (Karpen project, Pirrotta subgroup)Nuclei were isolated from the Drosophila BG3 neuronal cell line that had been transfected with dsRNA targeting the CP190 gene, and crosslinked with formaldehyde. After lysis and sonication the chromatin was immunoprecipitated with affinity-purified antibodies that recognize various chromosomal proteins. 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 DescriptionThe 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.
Release Date: 2011-09-03