title: GSM4263689: WT histone turnover rep2; Neurospora crassa; ChIP-Seq
library_construction_protocol: Cultures were grown in Vogels medium and 1.5% sucrose (standard minimal medium), shaking for 18 hours at 32oC in complete darkness. Cultures were spiked with 100mM hydroxyurea to block DNA replication and incubated for 3hrs. To induce hH3-3xFLAG expression, cultures were exposed to blue light (465 nm; 30 mole photons/m^2/s) for two minutes, and incubated for the indicated time to allow for incorporation into chromatin. Mycelia were washed in PBS prior to crosslinking. Mycelia were cross-linked for 30 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 20 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, 40µL of 50:50 anti-FLAG conjugated bead slurry was added, and samples were rotated overnight at 4oC. The next day, the beads were washed twice in cold ChIP Lysis buffer, once with ChIP Lysis buffer + 0.5M NaCl, once with LiCl 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 65˚C.  Samples (IP and input) were decrosslinked at 65˚C overnight.  The next day, samples were proteinase K treated for 2 hours at 50˚C, 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.
attributes.tag: GEO Accession
attributes.value: GSM4263689