ChIP-Seq IP
Fresh mycelia were cross-linked with 1% formaldehyde for 15 minutes, followed by quenching with 125 mM glycine. The cultures were ground in liquid nitrogen and resuspended in Nuclear Extraction Buffer (2 mM EDTA, 2.5 mM DTT, 10 mM HEPES (pH 8.0), 0.4 M sucrose, 1 mM sodium vanadate) supplemented with protease inhibitors (Beyotime, Shanghai, China). Chromatin was sheared to ~300 bp fragments using a XM-26A non-contact ultrasonic DNA interrupter (Jiangsu, China) with 40% amplitude for 30 cycles (30 sec ON and 30 sec OFF per cycle). After centrifugation, the supernatant was diluted with ChIP Dilution Buffer (16.7 mM Tris-Cl (pH 8.0), 1.2 mM EDTA, 1% Triton X-100, 167 mM NaCl, 1 mM PMSF, protease inhibitors). Immunoprecipitation was performed using either Rabbit anti-GFP-Tag mAb (ABclonal, Wuhan, China) or anti-Rabbit IgG (ABclonal, Wuhan, China). Protein A/G magnetic beads (ABclonal, Wuhan, China) were sequentially washed with: Low-salt wash buffer (2 mM EDTA, 20 mM HEPES (pH 8.0), 150 mM NaCl, 0.1% SDS, 1% Triton X-100), High-salt wash buffer (50 mM HEPES (pH 7.9), 500 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate), LiCl wash buffer (1 mM EDTA, 10 mM HEPES (pH 8.0), 0.25 M LiCl, 1% Nonidet P-40, 1% sodium deoxycholate), TE buffer (twice, 50 mM Tris-HCl (pH 8.0), 10 mM EDTA). DNA was eluted from the beads using freshly prepared Elution Buffer (1% SDS, 0.1 M NaHCO), followed by reverse cross-linking with 5 M NaCl, 0.5 M EDTA, 20 L 1 M Tris-HCl (pH 8.0), 2 L Proteinase K, and 3 L RNase A. DNA was purified using a Tiangen DNA purification kit (Tiangen, Beijing, China) or phenol-chloroform extraction. Further, ChIP-enriched DNA was sent for sequencing on Illumina (Annoroad Genetic Technology, Beijing, China)