GSM3560801: Control_H3K27Me3; Drosophila melanogaster; ChIP-Seq
Dechorionated embryos were fixed in 1:1 solution of 1.8% formaldehyde and heptane for 15 min at room temperature. The fixative was quenched with a 2 min wash with 125 mM glycine in PBS with 0.1% Triton-X100 (PBS:130 mM NaCl, 7 mM Na2HPO4, 3 mM KH2PO4, pH 8.0), and then briefly rinsed with PBS 0.1% Triton-X100. Embryos were snap frozen in liquid nitrogen, and stored at −80°C. About 1g of embryos of each genotype were used for nuclei isolation as described in Bowman et al. (2014, 2013). Fixed nuclei from wor>dsRed embryos were enriched using a Bio-RAD S3e cell sorter with 561nm excitation. Nuclei were sorted at 40C in 100ul of PBS, with 1% BSA, 0.1% Triton-X and 1X protease inhibitor. wor-gal4 is expressed in the nervous system from stage 11 onwards.  The sorted nuclei represent approximately 0.5-3% of total embryonic nuclei, and were at least 50% pure, based on post-isolation assessment of ds-red by confocal microscopy.  About a million nuclei were used for chromatin preparation. After isolation chromatin was fragmented with 15U of micrococcal nuclease (MNase, Worthington Biochemical) followed by 3 min sonication in a Diagenode Bioruptor 377.  Immunoprecipitation was carried out with 2ug of H3K27me3 antibody (Active Motif 39136) or 1ug of H3K27Ac antibody (Active Motif, 39136). Input DNA was used as a control.  Single end tag libraries were prepared and sequenced on an Illumina, Hiseq2500 in high output mode at the MGH Next Generation Sequencing Core.
GEO Accession
GSM3560801