Komagataella phaffii
Shaking cultured yeast cells were fixed by 1% formaldehyde for 15 min before harvested by centrifugation. Cells were washed by PBS twice and grounded in liquid nitrogen. 1.5 mL cell powder was suspended in 3 mL ChIP lysis buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.25% Triton X-100, 0.5% NP-40, 1mM PMSF), and sonicated to break chromatin into near 200 bp fragments. 1 mL supernatant was pre-cleared by 20 L blocked (5% BSA, 100ug/ml sheared salmon sperm DNA in ChIP buffer) protein G beads. After centrifugation again, 50 L supernatant was saved as input sample, and the rest was incubated with H3K36me3 or H3 antibody (Abcam, 25 g chromatin with 2-4 g antibody) at 4 overnight. 30 L blocked protein G beads were then added, followed by 4 incubation for 1-2 h. The beads were then washed at 4 C for 5 min with the following buffers: Low Salt Immune Complex Wash Buffer (10 mM Tris-HCl pH 8.0, 150 mm NaCl, 1 mM EDTA pH 8.0) once, High Salt Immune Complex Wash Buffer low salt buffer (10 mM Tris-HCl pH 8.0, 500 mm NaCl, 1 mM EDTA pH 8.0) once, LNDET buffer (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA) buffer once, and TE buffer (10 mM Tris-HCl pH 8.01 mM EDTA pH 8.0) twice. Both input and IP samples were then de-crosslinked by 0.2M NaCl at 65 water bath overnight, and DNA fragment were purified by the phenol-chloroform method. After elution, both input and pulldown samples were subjected to library construction (NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina) and high-throughput sequencing (Genewiz).