Cultures were processed as previously described (doi:10.1093/nar/gkz119), and the final pellet after cell lysis was resuspended in 500 μL LB140 and sonicated in a QSonica Q800R machine for 15 minutes (30 seconds on, 30 seconds off, 70% amplitude) to achieve 100-500 bp DNA fragments. Protein concentrations were measured by Bradford assay, and 1 mg of chromatin was used as input. As a spike-in control, S. pombe chromatin from a strain encoding an HA-tagged protein (FWP568) was added to a final concentration of 10%, and the volume was brought up to 800 μL with WB140 buffer. For immunoprecipitation, 10 μg anti-HA (Abcam, ab9110) was used. Immunoprecipitation reactions were incubated at 4°C with end-over-end rotation overnight (~16-18 hr). Protein G Sepharose beads (GE Healthcare, 4 Fast Flow) were washed with WB140, and a 50% slurry was subsequently made in WB140. Fifty-μL aliquots of slurry were transferred into new Eppendorf tubes, and the WB140 was removed. The immunoprecipitation reactions were added directly to the beads and incubated at 4°C with end-over-end rotation for 4 hr. Bead-washing and elution were done are previously described (doi:10.1093/nar/gkz119). The 200 μL of eluate was incubated at 65°C overnight to reverse crosslinking. RNase A/T1 digestion, Proteinase K digestion, and DNA purification were done as previously described (doi:10.1093/nar/gkz119). Library preparation was done as previously described (doi:10.1093/nar/gkz119). The number of PCR cycles for library amplification was determined empirically for each sample and ranged from 11-19 cycles. Samples were again purified twice using 0.7x volume SPRI beads and run on Agilent Bioanalyzer to assess size distribution, quality, and concentration.
GEO Accession
GSM4647445