GSM5856065: Spp1-FLAG_Input_asynch_rep2; Saccharomyces cerevisiae; ChIP-Seq
All ChIP and eSPAN assays were performed as described previously (Yu et al., 2014). For H3K4me3 and H3K56ac ChIP, chromatin was pelleted, washed twice with NP buffer (1.6 M sorbitol, 2 mM CaCl2, 5mM MgCl, 50 mM NaCl, 14 mM b-mercaptoethanol, 10 mM Tris-HCl (pH 7.4), 0.075% NP-40, 5 mM spermidine), resuspended in the same buffer, and digested with MNase (9013-53-0, Worthington Biochemical) for 20 min at 37C yielding a majority of mono- and di-nucleosome fragments. Digestion was stopped with the addition of 5 l 0.5 M EDTA, 1/5 volume of 5xChIP lysis buffer, and incubating 10 min on ice. For RNA Pol II CTD-Ser5p ChIP, chromatin was sheared by sonication using a Bioruptor Pico (15 seconds ON, 30 seconds OFF, 10 cycles)(Diagenode) to yield fragments of an average size of 300 bp.   Sonicated and MNase-digested chromatin was cleared by centrifugation and immunoprecipitated with the appropriate antibody (anti-H3K4me3 (ab8580, Abcam), anti-H3K56ac (38), or anti-RPB1 CTD-Ser5p (04-1572, Millipore)) and protein G Sepharose beads (17-0618-02, GE Healthcare). After washing the beads extensively, DNA from both input and ChIP was recovered with the Chelex-100 protocol (39) and used for BrdU IP/eSPAN or purified with MinElute PCR Purification kit (28004, Qiagen). Single-stranded DNA sequencing libraries were prepared using the Accel-NGS 1S Plus DNA library kit (10096, Swift Biosciences). Input and ChIP DNA was used for BrdU immunoprecipitation to prepare BrdU IP and eSPAN samples, respectively. Recovered DNA from the Chelex-100 extraction was incubated at 100C for 5 min and immediately chilled on ice for 5 min. BrdU immunoprecipitation was performed by diluting DNA 10-fold with BrdU IP buffer (PBS, 0.0625% Triton X-100(v/v), 6.7 g/ml Escherichia coli tRNA, 0.17 g/ml BrdU antibody (555627, BD Bioscience)) and incubated for 2 hours at 4C. Protein G Sepharose beads were added to each sample for 1 additional hour at 4C. Beads were washed extensively and DNA was recovered with TE + 1% SDS at 65C  for 15 min and purified with MinElute PCR Purification kit (28004, Qiagen). Single-stranded DNA sequencing libraries were prepared using the Accel-NGS 1S Plus DNA library kit (10096, Swift Biosciences). ChIP-seq libraries were prepared and deep sequencing was performed by Columbia University Genome Center.