Anti h3k4me3

FF lung tumor (LT; 3 mm³) samples consisting of almost 70% tumor tissue were pulverized under liquid nitrogen, fixed with 1% formaldehyde for 10 min, and immediately neutralized with 0.125 M glycine for 5 min. The resulting cells were washed with 1X PBS and treated with lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA pH 8, 1% Triton X-100, 0.1% sodium deoxycholate, and 0.1% SDS) containing 10 ml with protease inhibitors (complete Mini, Roche, Indianapolis, IN, USA).
The chromatin from the FF-LT samples was sonicated for 10 pulses of 20 seconds w/u 60 watts. Then, 10 μg of chromatin was immunoprecipitated (ChIP) using a commercial EZ-Magna ChIP™ G kit (Millipore, Temecula, CA, USA) and 2.5 μg of anti-MEOX2 (Santa Cruz Biotechnology, Dallas, TX, USA) as well as 1 μg of activated anti-H3K27Ac, anti-H3K4me3, anti-H3K27me3, anti-H3K9me3 or anti-RNA Pol II antibodies (Abcam, Cambridge Science Park, Cambridge, U.K.). A 1-μg aliquot of anti-mouse IgG was used as a negative control for ChIP (Millipore, Temecula, CA, USA).

ChIP-seq was performed as described previously^{19 (link),46 (link)}. Briefly, crosslinked chromatin was fragmented by Micrococcal nuclease digestion and immunoprecipitated with anti-H3K4me3 or anti-H3K27me3 antibodies (Abcam, Cambridge, MA). After ChIP, the crosslinking was reversed and DNA fragments were purified. The sequencing library was constructed using Illumina’s TruSeq sample preparation kit according to the provided instruction. The samples before immunoprecipitation served as input controls. Sequencing was performed using Illumina Nextseq 500 sequencer.

ChIP assays were performed on 14-day-old in vitro roots using anti-GFP (Clontech Cat # 632592), anti-IgG (Millipore), anti-H3K27me3 (Millipore), anti-H3K4me3 (Abcam) and RNA Polymerase II (Abcam) antibodies, modified from [26 (link)]. Briefly, after plant material fixation in 1% (v/v) formaldehyde, tissues were homogenized, nuclei isolated and lysed. Cross-linked chromatin was sonicated using a water bath Bioruptor UCD-200 (Diagenode, Liège, Belgium) (15s on/15s off pulses; 15 times). The complexes were immunoprecipitated with antibodies, overnight at 4°C with gentle shaking, and incubated for 1 h at 4°C with 50 μL of Protein AG UltraLink Resin (Thermo scientific). Immunoprecipitated DNA was then recovered using the IPure kit (Diagenode, Liège, Belgium) and analysed by quantitative real-time PCR. An aliquot of untreated sonicated chromatin was processed in parallel and used as the total input DNA control.