Genome-wide chromatin reconstitution with Drosophila embryo extract and nucleosome positioning by CG7372 and su(Hw)

We have reconstituted chromatin in vitro on a genome-wide level by incubating total genomic Drosophila melanogaster DNA in D. melanogaster preblastoderm embryo extract. We analyzed chromatin reconstituted with 1) extract from wildtype embryos, 2) extract from embryos lacking the chromatin remodeling enzyme subunit Acf , and 3) extract from Acf-mutant embryos supplemented with recombinant ACF (Acf + Iswi). As a comparison, we also reconstituted chromatin on genomic DNA by salt gradient dialysis. We determined nucleosome positions in reconstituted chromatin by MNase-digestion followed by paired-end sequencing of mononucleosomal fragments. We also determined nucleosome positions in D. melanogaster BG3-c2 cells depleted for the proteins su(Hw) and CG7372 by RNA interference. Chromatin was assembled in vitro on Drosophila genomic DNA either by incubating the DNA in Drosophila preblastoderm embryo extract or by salt gradient dialysis of purified histones onto the DNA. The assembled chromatin was digested with MNase to predominantly mononucleosomal fragments. DNA was purified and sequenced paired-end on an Illumina 1500 HiSeq machine. For mapping nucleosomes in Drosophila BG3-c2 cells, nuclei were isolated and chromatin digested with MNase. DNA was purified, run on an agarose gel, the mononucleosomal band cut out and sequenced paired-end on an Illumina 1500 HiSeq machine.