GSM8322185: mcm23A_H3K56ac_eSPAN_rep1; Saccharomyces cerevisiae; ChIP-Seq
All ChIP and eSPAN assays were performed as described previously (Yu et al., 2014). To carry out ChIP for H3K4me3 and H3K56ac, chromatin was pelleted and washed twice with NP buffer (1.6 M sorbitol, 5 mM MgCl2, 50 mM NaCl, 2 mM CaCl2, 14 mM β-mercaptoethanol, 0.075% NP-40, 10 mM Tris-HCl (pH 7.4), 5 mM spermidine). The chromatin was then resuspended in the same buffer and digested with 4U MNase (9013-53-0, Worthington Biochemical) for 20 minutes at 37°C, resulting in primarily mono- and di-nucleosome fragments. The digestion was terminated by adding 5 μl of 0.5 M EDTA and 1/5 volume of 5x ChIP lysis buffer, followed by incubation on ice for at least 30 minutes. Following MNase digestion, the chromatin was cleared by centrifugation and then immunoprecipitated using specific antibodies (anti-H3K4me3 (ab8580, Abcam) and homemade anti-H3K56ac along with protein G Sepharose beads (17-0618-02, GE Healthcare). The beads were extensively washed, and DNA from both Input and ChIP samples was extracted via the Chelex-100 protocol and utilized for BrdU IP/eSPAN or purified using the MinElute PCR Purification Kit (28004, Qiagen) for ChIP. BrdU immunoprecipitation was performed using either input DNA for BrdU-IP-ssSeq, and H3K4me3 or H3K56ac ChIP DNAs for eSPAN. Briefly, input or ChIP DNA obtained from the Chelex-100 extraction was incubated at 100°C for 10 minutes and then immediately cooled on ice for 10 minutes. BrdU immunoprecipitation was conducted by diluting the DNA ten-fold with BrdU IP buffer (PBS, 0.0625% Triton X-100 (v/v), 6.7 μg/ml Escherichia coli tRNA, 0.17 μg/ml BrdU antibody (555627, BD Bioscience)) and rotating for 2 hours at 4°C. Protein G Sepharose beads were added and incubated for another hour at 4°C. After extensive washing, the DNA was extracted with 100 μl of 1x TE buffer containing 1% SDS at 65°C for 15 minutes and then eluted again with 50 μl of 1x TE buffer containing 1% SDS. The collected supernatants were subsequently purified using the MinElute PCR Purification Kit (Qiagen, 28004). Quantitative PCR was performed to analyze Input, BrdU-IP, ChIP, or eSPAN DNA using primers for the early replication origin ARS607 and the distant site ARS607+14kb. Single-stranded DNA sequencing libraries were prepared using the Accel-NGS 1S Plus DNA Library Kit (10096, Swift Biosciences).