Western Blotting

We performed Western blots from acid-extracted histones, probing for H3K9me2, H3K9me3, H3K4me3, and total H3. Briefly, ∼30 flies of each karyotype were dissected on dry ice to remove the abdomen. The resulting heads and thoraces were ground in Phosphate-buffered saline (PBS) plus 10 mM sodium butyrate and were acid extracted overnight at 4 °C. Samples were then run on a 4–12% gradient bis-tris gel and transferred to a nitrocellulose membrane using Invitrogen’s iBlot Dry Transfer Device. After blocking with 5% milk in PBS, we incubated membranes overnight with either 1:1,000 H3K9me2 antibody (Abcam ab1220), 1:2,000 H3K9me3 antibody (Abcam ab8898), 1:2,000 H3K4me3 antibody (Abcam ab8580), or 1:2,000 H3 antibody (Abcam ab1791) in Hikari Signal Enhancer (Nacalai 02272). We then incubated membranes with 1:2,500 secondary antibody (Licor 68070 and 32213), imaged bands on a Licor Odyssey CLx Imager, and quantified intensity using ImageJ.