Anti trimethyl h3k4

ChIP experiments were performed as previously described (Saleh et al., 2008) with some modifications. A 3 g sample of leaves was fixed with cross‐linking buffer and 1% formaldehyde using vacuum infiltration two times for 15 min each, and the cross‐linking reaction was quenched with 0.125 m glycine. The leaves were ground in a mortar and pestle in liquid nitrogen, resuspended in nuclei isolation buffer and then filtered through Miracloth. After centrifugation, the pellets (nuclei) were resuspended in cold nuclei lysis buffer and sonicated (Bioruptor^® Plus sonication device, Diagenode, Belgium). After centrifugation, the supernatant was pre‐cleared with Magna ChIP™ Protein A Magnetic Beads (EMD Millipore corporation, Temecula, CA), and specific antibodies were added and incubated overnight at 4 °C. The specific antibodies used were as follows: anti‐H2Bub1 (Cell Signaling Technology, Danvers, MA) and anti‐trimethyl‐H3K4 (Abcam). The enriched DNA fragments were detected by RT‐qPCR and compared with the input samples, the amount of immunoprecipitated chromatin as normalized to the total amount of chromatin used in GhDREB P1 region in wild‐type plants was given as 1. GhUBI1 was used as a negative control.

Chromatin immunoprecipitation (ChIP) assays were performed as described previously (38 (link),39 (link)). Briefly, 5 million cells were fixed with 1% formaldehyde and sonicated for 180 s (10 s on and 10 s off) on ice with a Branson sonicator with a 2-mm microtip at 40% output control and 90% duty cycle settings. The sonicated chromatin (1 ml) was clarified by centrifugation, aliquoted and snap-frozen in liquid nitrogen. To perform ChIP, sonicated chromatin (150 μl) was diluted 10-fold and purified with specific antiserum (2–5 μl) and protein G-agarose (60 μl). ChIP antibodies were obtained from Abcam (Cambridge, MA), including anti-trimethyl-H3-K4 (#ab8580), trimethyl-H3-K9 (#ab8898) and trimethyl-H3-K27 (#ab24684). DNA that was released from the bound chromatin after cross-linking reversal and proteinase K treatment was precipitated and diluted in 100 μl of low-TE buffer (1 mM Tris, 0.1 mM EDTA). PCR reactions were performed under liquid wax in a reaction containing 1 μl ChIP (or input) DNA, 0.5 μM appropriate primer pairs, 50 μM deoxynucleotide triphosphate and 0.2 U Klen-Taq I (Ab Peptides, MO). Standard PCR conditions were 95°C for 2 min, followed by 32 cycles of 95°C for 15 s, 65°C for 30 s of annealing and 72°C for 30 s of extension. The PCR products were separated on a 5% polyacrylamide–urea gel and quantified by a Phosphorimager (Molecular Dynamics, CA).