Chromatin Proteins in ML-DmBG3-c2 cells

Chromatin-binding Proteins in ML-DmBG3-c2 cells (Karpen project, Pirrotta subgroup)

Details

Nuclei were isolated from the Drosophila BG3 neuronal cell line that had been crosslinked with formaldehyde. After lysis and sonication the chromatin was immunoprecipitated with an affinity-purified antibody that recognizes the large sub-unit of RNA polymerase II. 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 eaveraged experimental replicates were analyzed using a peak-calling algorithm that calculates genomic regions of significant enrichment (or depletion).

General Description

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.

Protocols

  1. Growth and isolation: Chromatin prep from cultured cells, Crosslinking of cultured cells, Crosslinking of cultured cells, Chromatin prep from cultured cells
  2. Sample preparation: Hybridization to Affy arrays, WGA, ChIP, Array Scanning Protocol
  3. Data Analysis: Regions of significant enrichment, Smoothed M-value enrichment profiles, M-value normalization, Smoothed M-value enrichment profiles, Regions of significant enrichment

Experimental Reagents

  1. Antibodies: Ab:POF_(MO_459):GK:1, HP1 wa191, SU(HW)-HB, Chro(Chriz)BR, Ab:HP2_(Ab2-90):GK:1, HP1c (MO 462), RNA pol II (ALG)
  2. Arrays: Affymetrix Drosophila Tiling Arrays v2.0R

Sample Details

  1. Animals/Lines: ML-DmBG3-c2


  1. Other Protocols: Regions of significant enrichment, Regions of significant enrichment


Release Date: 2009-10-12