Illumina Genome Analyzer IIx sequencing; Time course Pho/dSfmbt Chromatin Immunoprecipitation on Drosophila melanogaster embryos during embryogenesis
Time course Pho/dSfmbt Chromatin Immunoprecipitation on Drosophila melanogaster embryos during embryogenesis
Wildtype Drosophila melanogaster (Twist-H2B) flies were grown in population cages at 25 degr. Staged 2 hr populations of embryos were collected and aged at 25 degr till the required stage of development. Two independent collections were performed for each timepoint. The collected embryos were dechorionated using 50% bleach and formaldehyde fixed in 10 ml cross-linking solution (50 mM Hepes, 1 mM EDTA, 0.5 mM EGTA, 100 mM NaCl, 1.8 % formaldehyde, pH 8.0) and 30ml n-heptane on a shaker table at RT for 15 minutes. The reaction was terminated with 125 mM glycine, 0.1% Triton X-100 in PBS. After washing out the fix, the embryos were blotted dry and frozen in liquid nitrogen. A small number of embryos from each collections was set aside to stage each collection.  Solexa libraries were prepared according to manufacturers recommendations with small modifications.  In short, 3-10ng of IP-purified, RNase treated, and reverse cross-linked genomic DNA was end-repaired and terminal adenosine residues were added using the NEBNext reagents.  PE adapters (Illumina) were ligated, after which the material was size selected at ~230-250 bp (size equals sheered chromatin fragments plus adapters) on a 2% GelGreen (Biotium) stained agarose gel using SafeXtractor-100 gel extractors (5Prime) under blue light.  PCR amplification was performed using PE1.0 and PE2.0 primers (Illumina) for 18 cycles according to manufacturer's recommendation using the Phusion High-Fidelity PCR Kit (Finnzyme).  The PCR-amplified library was purified on a 2% agarose gel, avoiding unincorporated primers and primer-self-ligation products.  Library quality was assessed on a Bioanalyzer2100 system (Agilent).