Insulators ChIP-Chip

Insulators ChIP-chip (White project)

Details

Genetic material were isolated from Drosophila S2-DRSC cultured cells . This was cross-linked with formaldehyde. After lysis and sonication the chromatin was immunoprecipitated with antibodies recognizing the N-terminus of CTCF. The DNA was amplified, labeled, and hybridized to Affy microarrays (mock IP DNA was labelled in parallel and hybridized simultaneously to the same chip). Density plots are the combination of three data sets using MAT. The Joint Binding Deconvolution algorithm was used to predict binding peaks in normalized data from three experimental replicates.

General Description

The White Lab is aiming to map the association of all the Transcription Factors (TF) and DNA associated proteins on the genome of Drosophila melanogaster. The main technique that will be used for this purpose is chromatin immunoprecipitation-on-chip (ChIP-on-chip) utilizing whole-genome tiling arrays. The data generated by ChIP-chip experiments consist basically of a plot of signal intensity across the genome. The highest signals correspond to positions in the genome occupied by the tested TF.
ChIP-seq, meaning direct sequencing of the ChIP sample on the Solexa platform, has also been used to validate some datasets and will become- as the project advances- the main production platform.

To support ChIP-chip and ChIP-seq datasets, and in order to link association of factors to the DNA with regulation of transcription, several RNA-seq (direct sequencing of the transcriptome on the Solexa platform) datasets will be genrated for the time-points under scrutiny.

Protocols

  1. Growth and isolation: Embryonic collection Stage 0-12 hours AEL:KW:1, Cell Lines Chromatin Collection:KW:1
  2. Sample preparation: Whole_Genome_Amplification, Affymetrix Labeling and Hybridization:KW:1, White_Lab/IP, Array Scanning, Cross-linking, White_Lab/IP, Affymetrix Labeling and Hybridization:KW:1, Whole_Genome_Amplification
  3. Data Analysis: Data Processing:KW:1, Data Processing:KW:1
  4. Other Protocols: Cross-linking

Experimental Reagents

  1. Antibodies: No Antibody Control, CTCF-N, BEAF-32, Su(Hw), Su(Hw)-PG, CTCF-C, GAF, CP190, mod(mdg4)
  2. Arrays: Affymetrix Drosophila Tiling Arrays v2.0R, Affymetrix Drosophila Tiling Arrays v2.0R

Sample Details

  1. Animals/Lines: Embryo 0-12h, Kc167, Embryo 0-12h, S2-DRSC


Release Date: 2008-08-08