title: GcnE-FLAG_Srapa_noSiran_H3K9ac_Rep1
organism: Aspergillus nidulans
description: Protocols: A. nidulans was cultivated in Aspergillus minimal medium (AMM) at 37 °C and 200 rpm (41). AMM was supplemented according to the requirements of the strains as follows: arginine (50 µM), p-aminobenzoic acid (3 µg mL-1) or pyridoxine HCl (5 µg mL-1). Pre-cultures were inoculated with 4 × 106 spores mL−1. For measurement of orsellinic acid, mycelia of overnight cultures (~16 h) in AMM were transferred to fresh medium and inoculated with S. rapamycinicus, as described by Schroeckh et al. . RNA extraction for expression analysis during co-cultivation was performed after 3 h of incubation, samples for HPLC analysis were taken after 24 h. Cultures were essentially grown as described above and cross-linked after 3 h for 15 min with 1 % (v/v) formaldehyde at room temperature. The reaction was stopped by the addition of 125 mM glycine for 5 min at 37 °C and subsequently frozen in liquid nitrogen. Powdered mycelium was dissolved in 1 ml of sonication buffer and 330 µl aliquots were then subjected to sonication for 30 min with cycles of 2 min maximum intensity followed by a 1 min pause. Sheared chromatin was separated from cell wall debris and incubated with 40 µl of a protein A slurry for 30 min at 4 °C on a rotary shaker. A purified 1:10 dilution of the supernatant was then incubated overnight at 4 °C with 3 µl of antibody directed against the desired target. Antibodies were precipitated with 40 µl of dynabeads incubated with the sample for 40 min at 4 °C on a rotary shaker. Samples were washed 3 times with low salt followed by one time high salt buffer. Washed beads were dissolved in 125 µl TES buffer and reverse cross-linked by co-incubation with 2 µl of 0.5 M EDTA, 4 µl of Tris-HCl and 2 µl of 1 mg/ml proteinase K for 1 h at 45 °C. Subsequent DNA purification was conducted with a PCR purification kit and samples were eluted in 100 µl of 1:10 diluted elution buffer. Concentration of specific DNA was quantified using qRT-PCR essentially as described above. Antibodies for the experiments conducted are as follows: mouse monoclonal ANTIFLAG M2 (Sigma), rabbit polyclonal anti-histone H3 (Abcam), rabbit polyclonal Histone H3K9me3 (Active Motif) and rabbit polyclonal anti-acetyl-histone H3 (Lys14) (Millipore). Cutout size: 100-700, NEBNext Dual 705:ATTCAGAA + 505:AGGCGAAG