Chromatin Silencing ChIP-chip arrays

Chromatin Silencing enzymes (Lieb project, Lieb subgroup)

Synchronized C. elegans mixed stage embryos from strain N2 were treated with the cross-linking reagent formaldehyde. Sonicated chromatin was prepared and immunoprecipitated with an affinity-purified polyclonal antibody that recognizes essential DOT1 methylase domain protein Y39G10AR.18. After whole genome amplification, NimbleGen genomic tiling microarrays were used in two-color hybridization experiments to compare the signal from the input DNA versus the fragments pulled-down in the ChIP. Normalized log2(ratios) are shown in this track.



General Description

The focus of our analysis will be elements that specify nucleosome positioning and occupancy, control domains of gene expression, induce repression of the X chromosome, guide mitotic segregation and genome duplication, govern homolog pairing and recombination during meiosis, and organize chromosome positioning within the nucleus. 126 strategically selected targets include key histone modifications, histone variants, RNA polymerase II isoforms, dosage-compensation proteins, centromere components, homolog-pairing facilitators, recombination markers, and nuclear-envelope constituents.
We will integrate information generated with existing knowledge on the biology of the targets, perform ChIP-chip analysis on mutant and RNAi extracts lacking selected target proteins, use extrachromosomal arrays to assess the ability of candidate identified sequence motifs to recruit targets in vivo, identify tissue-specific patterns of selected targets, and create integrated, quantitative models of transcription and whole-chromosome functions.

Protocols

  1. Growth and isolation: Worm popcorn chromatin extract protocol vSS1:JL:1, Worm embryo growth and harvest:JL:5, Worm embryo growth and harvest:JL:SS7, Worm embryo extraction:JL:2, Worm embryo extraction:JL:3
  2. Sample preparation: ChIP-chip scanning nimblegen:JL:1, ChIP-chip label hyb nimblegen:JL:1, Worm LM-PCR Amplification for ChIP-chip:JL:SS2, ChIP-chip scanning nimblegen:JL:2, Worm Chromatin Immunoprecipitation:JL:MBJ5, Worm LM-PCR Amplification:JL:MBJ1, Worm Chromatin Immunoprecipitation:JL:SS4, ChIP-chip label hyb nimblegen:JL:2
  3. Other Protocols: ChIP-chip normalization standard MA2C:JL:1, ChIP-chip peak finding over reps MA2C:JL:1, ChIP-chip peak finding MA2C:JL:1, ChIP-chip normalization standard MA2C:JL:2


Release Date: 2010-06-21