GSM6935285: DMSO_H2Bub_IP_Rep1; Saccharomyces cerevisiae; ChIP-Seq
ChIP was performed essentially as described previously (70,71). Briefly, 200 or 250 mL of cell cultures were grown to log phase (OD600 = 0.8 - 1.0). Cells were crosslinked for 20 min at room temperature by addition of formaldehyde to 1% (v/v). Lysates were prepared in FA lysis buffer (72). Chromatin was sheared in Bioruptor® Pico 15 mL tubes with 300 µL beads (Diagenode C30010017) by 25 cycles of 30 sec on and 30 sec off using a Bioruptor® (Diagenode B01060010). Sonicated chromatin was brought up to a volume of 6 mL in FA lysis buffer and centrifuged at 50,000 rpm for 20 min at 4˚C. Chromatin in the supernatant was aliquoted, flash-frozen, and stored at -80˚C. S. cerevisiae chromatin was mixed with Schizosaccharomyces pombe chromatin (KP07 or KP08) at a 1:10 ratio (S. pombe : S. cerevisiae) based on protein concentration (Pierce BCA protein assay kit; Thermo Fisher Scientific 23227), and IP reactions were conducted with anti-V5 (Invitrogen R960-25, 1 µL/225 µg or 10 µL/1800 µg), anti-Rpb1-CTD (8WG16; BioLegend 664906, 1 µL/225 µg or 8 µL/1800 µg), anti-HSV (Sigma-Aldrich H6030, 1.75 µL/450 µg or 7 µL/1800 µg), anti-HA (Santa Cruz sc-7392 AC, 15 µL/225 µg), anti-Spt5 (gift from Grant Hartzog, 4 µL/1800 µg), anti-H2BK123ub (Cell Signaling 5546, 5 µL/1000 µg), anti-total H3 (Abcam ab1791, 2 µL/1000 µg), anti-H3K4me2 (Millipore 07-030, 5 µL/1000 µg), anti-H3K4me3 (Active motif 39159, 5 µL/1000 µg), or anti-Myc (gift from John Woolford, used as a non-specific IgG control, 5 µL/1000 µg) primary antibodies. While S. pombe chromatin was included for the ChIP assays in Fig. S4, it was not used for normalization of the S. cerevisiae data as it increased variance in the qPCR measurements. IP reactions were incubated overnight at 4˚C on an end-over-end roller. Protein A or G beads (Cytiva 17-5280-01 or 17-0618-01) were washed with FA lysis buffer with 275 mM NaCl and added to the chromatin-antibody mixture and placed on the roller for 1 hr at room temperature. Beads were washed and DNA recovered as previously described (71). DNA was purified using the QIAquick PCR Purification Kit (Qiagen, 28106). ChIP-sequencing (ChIP-seq) libraries were prepared using a NEBNext Ultra II kit and indices (E7645, E7335, E7500, E7710, E7730). Libraries were quantified by Qubit and fragment length distribution was assessed by agarose gel electrophoresis. DNA sequencing was conducted by the University of Pittsburgh Health Sciences Sequencing Core at UPMC Children's Hospital of Pittsburgh on an Illumina NextSeq2000.