Chromatin Proteins in Kc167 cells

Chromatin-binding Proteins in Kc167 cells (Karpen project, Karpen subgroup)

Nuclei were isolated from the Drosophila Kc167 embryonic cell line that had been crosslinked with formaldehyde. After lysis and sonication the chromatin was immunoprecipitated with an affinity-purified antibody that recognizes the cohesin complex protein Chromosome-associated protein. 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

We aim to determine the locations of 125 chromosomal proteins across
the Drosophila melanogaster genome. The proteins 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:HP1b_(Henikoff):GK:1, Ab:HP2_(Ab2-90):GK:1, Chro(Chriz)BR, HP1c (MO 462), RNA pol II (ALG)
  2. Arrays: Affymetrix Drosophila Tiling Arrays v2.0R

Sample Details

  1. Animals/Lines: Kc167


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


Release Date: 2010-02-15