GSM2264174: Ncrassa_N'CHAP_DNAaffinity; Neurospora crassa; ChIP-Seq
Amylose resin (NEB) containing immobilized MBP-CHAP was incubated with sonicated wild-type genomic DNA (~250 bp fragments) in binding buffer [20 mM HEPES (pH 7.5), 80 mM NaCl, 37.5 mM Imadazole, 0.7 mM MgCl2, 0.35 mM EDTA, 0.7 mM DTT and 17.8% glycerol] for 2 h at 4 degrees. Beads were washed 6 times with 1 ml of wash buffer [10 mM Tris (pH 7.5), 50 mM NaCl, 1 mM MgCl2, 0.5 mM EDTA and 4% glycerol]. Following washes, DNA was eluted with TES [20 mM Tris (pH 8.0), 10 mM EDTA and 1% SDS] by heating for 10 min at 65 degrees. Eluted samples were treated with proteinase K and DNA was purified with MinElute columns (Qiagen). Purified DNA was prepared for sequencing using Illumina TruSeq ChIP-Seq Sample Prep Kit.