Instrument: NextSeq 500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: RNA-seq: Mycelia were harvested and added to a tube containing ~350uL glass beads (260-300 micrometer, acid washed [Sigma Aldrich] and resuspended in sdH2O), 350uL NETS buffer (10mM Tris pH 7.5, 300mM NaCl, 1mM EDTA, 0.2% SDS) and 350uL phenol:chloroform:isoamyl alcohol (25:24:1) and lysed by a bead beater for 3 minutes at room temperature. Samples were cleared by centrifugation, and the aqueous (top) phase was mixed with 350uL of chloroform. After centrifugation, the aqueous phase was divided into two tubes containing 650uL cold 100% EtOH. Samples were precipitated at -20oC, pelleted by centrifugation, washed twice with 1mL 70% EtOH, and resuspended in DEPC-treated dH2O. RNA was treated with DNase (DNA-free™ DNA Removal Kit, Thermo Fisher Scientific) and cleaned (Agencourt® RNAclean XP® beads, Beckman Coulter). ChIP-seq: Mycelia were washed in PBS prior to crosslinking. Mycelia were cross-linked for 10 min in 0.5% formaldehyde for histone modification ChIP. Tissue added to ChIP lysis buffer (50mM Hepes pH 7.5, 90mM NaCl, 1mM EDTA, 1% Triton + proteinase inhibitors) and was 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 histone modification antibody was added (typically 2uL) and samples were rotated overnight at 4oC. The next day, equilibrated Protein A or Protein G slurry (agarose or magnetic beads) 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. Whole-genome-seq: Used input DNA from ChIP experiments. RNA-seq: RNA-seq libraries were prepared (KAPA Stranded mRNA-seq kit, KAPA Biosystems). ChIP-seq: Approximately 10 ng of DNA was used to generate ChIP-Seq libraries. Each library was prepared using the Illumina Tru-Seq kits A and B (Illumina, IP-202-1012 and IP-202-1024) 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. Whole-genome-seq: Performed library prep same as ChIP-seq samples.