Qiaquick dna purification columns

ChIP assay was conducted applying the SimpleChIP® enzymatic chromatin IP kits (Cell Signaling Technology, Danvers, MA, USA). Chromatin was incubated with antibodies against H3K9me3 (ab8898, Abcam), or JMJD2C (PA5-23065, Thermo Fisher Scientific), or IgG (serving as negative control, ab172730, Abcam) for immunoprecipitation. Eventually, the compound of immunoprecipitated protein and DNA was extracted and used for RT-PCR to confirm the binding site. The extraction of DNA was conducted as follow: samples were rinsed in the lysis buffer twice, and were rinsed in 1 M lysis buffer (50 mM Tris, pH 7.4, 1 M NaCl, 1 mM EDTA, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate) for 4 times, and were resuspended in lysis buffer, followed by 45 min treatment with proteinase K at 45°C. Co-immunoprecipitated DNA was purified by QIAquick DNA purification columns (Qiagen, Germantown, MD, USA) and were eluted using 50 μL nuclease-free water. The primers of the ALKBH5 promoter were as follows: forward primer: 5’-TCTCCTTTAGGGGTCCTCGC-3’, reverse primer: 3’GGAGTTTCCGGAAGTCGGTT-5’.

FAIRE was performed as described (Omidbakhshfard et al., 2014 (link)). For inflorescences, 0.3 g of tissue was crosslinked with 1% formaldehyde in crosslinking buffer under vacuum for 8 min. For tests in plant cells, 1 × 10⁶ protoplasts were crosslinked in 1% formaldehyde, 1x PBS for 8 min. Isolated DNA fragments were further purified by Qiaquick DNA purification columns (Qiagen, Germantown MD, United States). The Ta3 retrotransposon (At1g37110) (Johnson et al., 2002 (link)) was used as the NC locus for all FAIRE experiments. qPCR was performed for crosslinked and noncrosslinked FAIRE samples. Fold enrichment was obtained by normalizing DNA accessibility in FAIRE samples over that of un-crosslinked DNA. The fold enrichment at each experimental locus was normalized over that of Ta3.