Ab1761

For salivary gland polytene chromosome immunostaining of late L3 larvae, samples were incubated in PBS buffer with 3.7% paraformaldehyde and 45% acetic acid. Non-specific binding sites were blocked for 1 h at 25°C in 5% fetal calf serum (Lonza) or 5% BSA (Sigma-Aldrich) containing PBST (PBS + 0.1% Tween20). Then slides were incubated overnight at 4°C with anti-histone H3K9ac (Abcam ab4441, 1:200), anti-histone H3K14ac (Upstate 07-353, 1:200), anti-histone H4K12ac (Abcam ab1761, 1:200), anti-histone H4K8ac (Abcam ab1760, 1:100) anti-dADA2b (1:100) and anti-RNAPII (1BP-7G5, 1:500) [21 (link), 34 (link)] antibodies. Polyclonal rabbit antibodies against dTAF10 and dTAF10b were generated at IGBMC [41 (link)]. dTAF1, dTAF5 and dTBP antibodies were a kind gift from Y. Nakatani. Samples were incubated for 1 h at 25°C with secondary antibodies (Dylight 549, Alexa Fluor 555, and Alexa Fluor 488 IgGs, Molecular Probes). Slides were covered with Prolong Gold and examined with an Olympus BX51 microscope attached to a DP70 camera.

DNA was immunoprecipitated as described earlier [53 (link)] using 2μl of anti-H4K12Ac (ab1761, abcam), 2μl of anti-myc (9E10, Sigma), 1 μl of anti-GFP (ab290, abcam), or 3μl anti-RNA polymerase II CTD repeat (ab5408, abcam) antibodies per 100μl chromatin extracts.
For microarray hybridization, immunoprecipitated DNA was amplified to 5 μg DNA as described in [53 (link)], with the exception that in the second PCR, 5 mM dUTP was added to the reaction. Fragmentation, labeling and hybridization to the Affymetrix GeneChip S. pombe Tiling 1.0FR was performed by the Affymetrix core facility at Novum (http://apt.bea.ki.se) according to Affymetrix standard protocols.
All experiments were done as biological duplicates.
For real-time quantitative PCR, immunoprecipitated DNA was amplified in the presence of SYBR Green using Applied Biosystems 7500 real-time PCR machine. The primers used are listed in S2 Table.