title: GSM7136577: YRP10_Pdr1_IgG_rep2; Saccharomyces cerevisiae; OTHER
library_construction_protocol: CUT&RUN assays were carried out using the Epicypher CUTANA ChIC/CUT&RUN Kit (Epicypher 14-1048). Briefly, 1 million nuclei were thawed, immobilized on concanavalin A beads, and permeabilized in 0.05% digitonin cell permeabilization buffer. For each sample the permeabilized nuclei were divided equally and incubated in 50 µL antibody buffer with either 0.5µg of control rabbit IgG antibody (Epicypher 14-1048) or anti-c-Myc antibody (ThermoFisher Scientific 13-2500) overnight at 4°C. After washing the beads, pAG-MNase (EpiCypher 14-1048) and 100mM CaCl2 was added to the immobilized cells and incubated for 2h at 4°C to digest chromatin DNA. DNA cleavage was terminated by addition of 150 µl of stop buffer and 0.5 ng of E. coli Spike-in DNA (EpiCypher 14-1048). After incubation for 10m at 37°C, the CUT&RUN-enriched DNA was phenol-chloroform precipitated with 80 µg glycogen. The DNA was dissolved in 0.1x TE buffer, followed by DNA quantification using the Quant-iT PicoGreen assay (Thermofisher Scientific P7589). CUT&RUN libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB E7645S) following the manufacturer's instruction with modifications to SPRI bead size selection and library amplification reactions to retain small DNA fragments