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. Fixed mesodermal nuclei were isolated and used subsequently to perform chromatin immunoprecipitation as previously described by Bonn and colleagues (Cell type-specific chromatin immuno precipitation from multicellular complex samples using BiTS-ChIP, Nat Prot, 2012).  Briefly, whole embryos were homogenized and pushed through the needles to extract nuclei. Nuclei were stained with alpha-SBP antibody generated in mouse that recognizes SBP-tagged histone H2B.   Subsequently, nuclei were stained with secondary antibody alpha-mouse Alexa Fluor 488, which was used to separate mesodermal fixed nuclei using Fluorescence Activated Cell Sorting (FACS) with purity greater than 95%.   Several sorts were pulled together to obtain sufficient amount of material. Chromatin was sheared to 200 bp with Bioruptor and used to perform imunoprecipitation (IP) as previously described in Sandmann et al. (Nat Prot, 2006) with antibodies (generous gifts from Joerg Mueller recognizing Pho (2-382 aa) on 4-6h and 6-8h, or dSfmbt (531-980 aa) on 6-8h material.  The Extraction is part of IP described in the sample's ChIP protocol NGS libraries were prepared as previously described by Bonn and colleagues (Cell type-specific chromatin immuno precipitation from multicellular complex samples using BiTS-ChIP, Nat Prot, 2012).