Chromatin Proteins in CME-W1-Cl.8+ cells

RLPII_phospho2_ab5095.Clone_8.Solexa (Karpen project, Kuroda subgroup)

Nuclei were isolated from the Drosophila CME-W1-Cl.8+ wing imaginal disc 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 subunit of RNA polymerase II phosphorylated on heptapeptide serine 2 of its CTD. 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.

Details

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 the Illumina NGS platform. 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: Crosslinking of cultured cells, Chromatin prep from cultured cells
  2. Sample preparation: ChIP, Illumina sequencing
  3. Data Analysis: Bowtie alignment
  4. Other Protocols: SPP Peak calling

Experimental Reagents

  1. Antibodies: RNA polymerase II CTD repeat YSPTSPS (phospho S2)

Sample Details

  1. Animals/Lines: Drosophila melanogaster


Release Date: 2013-03-19