GSM7497702: mycelia - H3K27me3_ash1set2_rep2_IP; Pyricularia oryzae; ChIP-Seq
The ChIP experiments with mycelia cultured in liquid CM for 2 days were conducted as previous reports with minor modification (He et al., 2018; Tao et al., 2017). Briefly, 1.0 g mycelia were crosslinked with 1% formaldehyde for 20 mins and stopped with 125 mM glycine for 5 mins at room temperature. Samples were ground with liquid nitrogen and resuspended in nuclei isolating buffer. Subsequently the precipitated nuclei were used to total chromatin extraction with 1mL lysis buffer. The lysis chromatin was sonicated into DNA fragments between 200-500 bp using Diagenode Bioruptor. 20μL chromatin was used to input DNA extraction and the remainder was pre-cleared with 10μL protein A Dynabeads (Thermofisher, 10001D) for 1 hour. Subsequently the chromatin was incubated with anti-H3K27me3 (Abcam, ab6002) or anti-H3K4ac (Active Motif, 39381) overnight at 4°C. Another 20μL protein A Dynabeads was used capture protein-DNA mixture and followed by three washing. Protein-DNA mixture was reverse-crosslinked, and DNA was recovered with phenol-chloroform extraction. The recovery DNA was used as template for followed ChIP-qPCR and ChIP-seq. Two biological repeats were conducted. The purified DNA was used as libraries construction with the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645L).