GSM8141206: H3K4me3_ChIP mcm2_G221V rep2; Schizosaccharomyces pombe; OTHER
Cells were crosslinked at 60 minutes after temperature shift with the addition of 1% formaldehyde and incubated for 20 minutes with shaking at 25°C. Immunoprecipitation was performed as described in ChIP. Crosslinking was reversed by Chelex-100. Briefly, total and ChIP DNA were incubated at 100°C for 5 minutes and then immediately cooled on ice for 5 minutes. DNA was diluted ten times with BrdU IP buffer (PBS, 0.0625% Triton X-100(v/v)), and incubated with BrdU antibody (BD Bioscience 555627) for two hours at 4°C. Next, Sepharose Protein G beads (Cytiva 17-0618-01) were added and incubated for an additional one hour at 4°C. The beads were then washed three times with BrdU IP buffer and once with TE. Finally, DNA was eluted with TES and purified with the QIAGEN MinElute PCR Purification kit (Qiagen 28004). Accel­NGS 1S Plus DNA library kit (Swift Bioscience, 10096).