In vivo genomic binding sites for the Drosophila CTCF Insulator

Chromatin immunopurification with anti-CTCF was used to identify in vivo binding sites for CTCF in approximately 3% of the Drosophila genome using amplicon-based tiling arrays. This includes the Bithorax-complex, a known site of CTCF activity, the Antp-Complex, the Achaete scute-Complex and the Adh region. Reproducible binding identified new CTCF sites in the Adh region and the Bx-C. Keywords: ChIP-chip Formaldehyde-crosslinked Chromatin was prepared from 4 independent biological replicates (Embryo1, Embryo2, Embryo5 and Embryo6) of 0-20hr wild-type Drosophila embryos (OregonR strain). Each chromatin sample was immunopurified with rabbit Anti-CTCF and, in parallel with normal rabbit serum. DNA purified from the experimental and control immunopurifications was amplified by LM-PCR. Four seperate aliquots (Technical replicates) of each PCR reaction were labeled and hybridised to independent tiling array slides, comparing experimental and control on each slide. 2 slides were dye swapped compared to the other two. Each of the 4 technical replicates were normalised (VSN) and the average ratio of Experimental v control signals calculated. The average ratio for each of the 4 biological replicates was assessed for statistical significance using CyberT to calculate an mean ratio and p-value.