Flies of all six species were raised at 25C and 60% humidity on corn syrup based food. Two to four days post enclosure flies were moved to a plexiglas cage and fed yeast paste on 150mm apple agar plates. All collections resulted in embryos 2-4 hours in age (For D. pseudoobscura, opuntia powder was also sprinkled on apple agar plates to increase numbers of eggs laid). Plates were left in cages for 2 hours allowing flies to lay eggs on them. Afterwards plates were removed and allowed to incubate for an additional 1.5h at 25C till harvest started, at which point bleach was added to the plate for two minutes to dechorionate embryos. Collection windows for most species were 2-4 hours post egg laying to match D. melanogaster embryonic development. We observed a delay in D. pseudoobscura, and acceleration in D. simulans embryonic development, which are consistent with an earlier study about heterochrony in Drosophila embryonic development (PMID:10618397). Therefore, for these two species, collection windows were adjusted empirically based on two criteria: a. to ensure majority of embryos were within Bownes stage 5-8; b. to obtain optimal signal/noise ratio from ChIP-seq experiments. Collection plates were removed after 2 hours of egg laying and were incubated for additional 0.5, 1.5, or 2.5 hours at 25C until the start of harvest for 1-3, 2-4, or 3-5 hour collections, respectively. Embryos were fixed using a modified protocol from Furlong et.al. Briefly, embryos were transferred to a tube containing PBT (PBS with 0.1% Triton) and allowed to settle. After PBT being removed, embryos were crosslinked by being vortexed in a mixture of fixing buffer (50 mM HEPES, pH 7.5, 1 mM EDTA, 0.5 mM, EGTA, 100 mM NaCl, 1.8% formaldehyde) and heptane for 15 minutes at room temperature. To stop crosslinking, fixing solution and heptane were removed, and 2.5 M glycine in PBT was added to fixed embryos before they were vortexed for 1 minute. Embryos were washed with PBT and either used for preparation of whole cell extract for chromatin immunoprecipitation or rapidly frozen in liquid nitrogen and stored at -80C. For staging embryos, some fixed embryos were vortexed in methanol:heptane (V:V=1:1) for 10 minutes for devitellinization, and washed twice by methanol before being stored in methanol at -20C. ChIPs were done using a modified protocol from Furlong lab (http://furlonglab.embl.de/methods/extras/ChIP-on-chip-Drosophila.pdf). On the day before ChIP, 100 ul protein A-conjugated Dynobeads (Invitrogen 100-02D) were washed three times by block solution (PBS with 5 mg/ml BSA), and incubated with 20 ul affinity purified rabbit polycolonal anti-Drosophila melanogaster Twist antibody (a generous gift from Levine lab and Furlong lab) at 4C overnight. To prepare nuclear extract, embryos cross-linked in 1.8% formaldehyde were resuspended in buffer A1 (15 mM HEPES, pH 7.5, 15 mM NaCl, 60 mM KCl, 4 mM MgCl2, 0.5 % Triton X-100, 0.5 mM DDT, freshly added complete EDTA-free protease inhibitor cocktail (Roche 11873580001)), and homogenized six times by both pestles on ice. Nuclei were precipitated by centrifuge, washed three times by buffer A1, and resuspended in buffer A2 (15mM HEPES, pH 7.5, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1 % Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 0.5% N-lauroylsarcosine, freshly added complete EDTA-free protease inhibitor cocktail (Roche 11873580001)). Chromatin was fragmented by sonication to the average size of 400-500 bp, and the soluble fraction (whole cell extract, WCE) was precipitated by centrifuge. For each ChIP, 300 ul WCE (100-120 mg embryos) was incubated with pre-treated protein A-conjugated Dynobeads overnight at 4C by rotating. Beads were washed four to five times by RIPA buffer (50 mM HEPES, pH 7.5, 1 mM EDTA, 0.7% Na deoxycholate, 1% IGEPAL CA-630, 0.5 M LiCl), and bound chromatin was eluted by 100 ul elution buffer (50 mM Tris + 1% SDS) on a thermomixer at 65C for 20 minutes with agitation. Supernatant was transferred and 100 ul TE was added to each ChIP sample before reverse crosslinking. For control, 50 ul WCE was mixed with 50 ul TE and 100 ul elution buffer. Reverse crosslinking was done by incubating samples at 65C for six hours. Reverse crosslinked DNA was then first treated with RNase A (0.2 ug/ul) and then with proteinase K (0.2 ug/ul) for two hours at 37C and 55C, respectively. DNA was cleaned with phenol (Sigma P4557) and phenol-chloroform-isoamyl alcohol mixture (Sigma 77617). For precipitation, glycogen and NaCl were added to each sample to the final concentration of 0.15 ug/ul and 200 mM, respectively. DNA was precipitated by 2.5 fold (v:v) 100% ethanol at -80C for at least two hours before DNA being precipitated by centrifuge and washed with 70% ethanol at 4C. Precipitated DNA was solved in TE. Level of Twist enrichment was monitored by real-time PCR. For D. melanogaster, regions within the enhancers of brinker and rhomboid genes were chosen as positive controls, while a region in chromosome 2L as negative control (NonG). Similarly, for individual non-melanogaster species, a region upstream of tup gene, and regions within the open reading frame of the othologues of D. melanogaster Dscam gene were chosen as positive control, while orthologous regions of NonG was used as negative control. Sequences for oligos used in PCR are available upon request.