GSM4185068: ∆pas N7999 H3K27me2/3 ChIP-seq; Neurospora crassa; ChIP-Seq
Cultures were grown in Vogels medium and 1.5% sucrose (standard minimal medium), shaking for 18 hours at 32oC. Mycelia were washed in PBS prior to crosslinking. Mycelia were cross-linked for 10 min in 0.5% formaldehyde, glycine (125mM final concentration) was added for 5 minutes and tissue was washed with PBS.  Tissue was added to ChIP lysis buffer (50mM Hepes pH 7.5, 90mM NaCl, 1mM EDTA, 1% triton + proteinase inhibitors) and disrupted by sonication for thirty pulses before chromatin was sheared using a Bioruptor (Diagenode) for 15 min with a cycle of 30 sec on followed by 30 sec off, at high power. Lysates were cleared by centrifugation, 1/20th Input was saved, and H3K27me2/3 antibody was added (typically 2uL) and samples were rotated overnight at 4oC. The next day, equilibrated Protein A/G agarose (Santa Cruz Biotechnology) was added to bind the antibody, incubated for 3 hours at 4oC, and washed twice in cold ChIP Lysis buffer, once with ChIP Lysis buffer + 0.5M NaCl, once with LiCl was buffer (10mM Tris pH 8.0, 250mM LiCl, 1mM EDTA, 0.5% NP40), and DNA/protein was eluted with TES buffer (50mM Tris pH 8.0, 10mM EDTA, 1% SDS) and incubated at 65oC.  Samples (IP and input) were decrosslinked at 65oC overnight.  The next day, samples were proteinase K treated for 2 hours at 50oC, and DNA was purified using the QIAquick PCR purification kit and eluted in 30 μl of water.  Approximately 10 ng of DNA was used to generate ChIP-Seq libraries.  Each library was prepared using the NEB Next DNALibrary Prep Kit for Illumina according to the manufacturer's instructions.  “Invisible” fragments between 250-400 bp were excised and purified using the MinElute gel extraction kit (Qiagen, 28606).  Final libraries were PCR-amplified using one cycle at 98 °C for 30 sec, 10 cycles at 98 °C for 10 sec, 60 °C for 30 sec and 72 °C for 30 sec and a final extension at 72 °C for 5 min.