GSM1815459: WT_GAF; Drosophila melanogaster; ChIP-Seq
Lysates were prepared from sonicated nuclei and GAF-DNA complexes were isolated with antibody.  The GAF antibodies were custom-made by this lab while anti-RNA Pol II antibodies were provided by the lab of Dr. John T. Lis. ChIP-seq libraries were prepared using the Illumina ChIP-seq Library preparation Kit following the manufacture’s instructions with minor modifications. Briefly 10ng of purified ChIP DNA was end repaired by conversion of overhangs to phosphorylated blunt ends. A’ bases were added to the 3’ends of the DNA fragments and Illumina adapters (1:30 dilution) were ligated to the ends of ChIP fragments.  This fragment range corresponds to a ChIP fragment range of about 200 -300bp. Size selected fragments were PCR amplified for 12 cycles . Libraries were quantified and validated using Agilent Technologies 2100 Bioanalyser for size, concentration and purity. Q-PCR was repeated to confirm retention of relative enrichment.