GSM8603443: H3K4me3_rtf1_8_9A_bio4; Saccharomyces cerevisiae; ChIP-Seq
Cell pellets were resuspended in 1xFA buffer containing 0.5% SDS and 1 mM PMSF. Cells were lysed with acid-washed glass beads using 15 cycles of vortexing at maximum speed (30s on, 30s rest on ice). Lysate was separated from beads via syringe (10 mL, with P200 tip as filter), transferred to an ultracentrifuge tube (Beckman Coulter, #355603, Brea, CA), and centrifuged at 50,000 rpm for 20 min at 4°C in an ultracentrifuge (Beckman Coulter, Optima XPN-100). The supernatant was decanted, and the pellet was resuspended in 8 mL 1xFA buffer containing 0.1% SDS and 1 mM PMSF. The resuspension was spun in the ultracentrifuge as above, and the supernatant decanted. The resulting chromatin pellet was resuspended in 1.5 mL 1xFA containing 0.1% SDS and 1 mM PMSF and transferred to a 15 mL Bioruptor tube with sonication beads (Diagenode, C30010017). Chromatin  was sheared for 25 cycles (30s on and 30s off) at 4°C in a Bioruptor Pico (Diagenode, B01060010). Chromatin was transferred to an ultracentrifuge tube and 4.2 mL of 1xFA buffer containing 0.1% SDS and 1 mM PMSF was added. Chromatin was centrifuged at 50,000 rpm for 20 min at 4°C and the supernatant was retained, aliquoted, and flash frozen in liquid nitrogen. Chromatin aliquots were stored at -80°C.  S. pombe chromatin from strain KP08 was spiked-in to S. cerevisiae chromatin at a 1:9 protein ratio upon thawing of sheared chromatin. Chromatin was quantified using the Pierce BCA kit (Thermo Scientific, 23225). IPs were performed with 500 µg (for histone IPs) or 1000 µg (for all other IPs) spiked-in chromatin and samples were diluted to 700 µL or 1.4 mL in 1xFA buffer containing 0.1% SDS and 1 mM PMSF, respectively. ChIPs were performed with antibodies against the following epitopes/proteins: HSV (Sigma-Aldrich, H6030; 5 µl per 1000 µg), HA (Santa Cruz, HA-probe Antibody (F-7) AC, sc-7392; 60 µl per 1000 µg), Rpb1 (8WG16; BioLegend, 664906; 4 µL per 1000 µg), H3 (Abcam, ab1791; 1µL per 500 µg), H3K4me3 (Active Motif 39159, 39160; 2.5 µl per 500 µg), and H3K36me3 (Abcam ab9050: 6 µg per 500 µg). After overnight incubation of primary antibodies with chromatin, Protein A Sepharose beads (Cytiva, 17-5280-01) were added to HSV, 8WG16, and H3K4me3 reactions in an amount of 30 µL beads (50% slurry) per 500 µg chromatin. Protein G Sepharose beads (Cytiva, 17-0618-01) were added to H3 and H3K36me3 in an amount of 30 µL beads (50% slurry) per 500 µg chromatin. Samples with Protein A or G beads were rotated end-over-end for 2 hr at 4°C. IP reactions were performed as described above for ChIP-qPCR, with input samples performed in parallel. DNA was eluted in 100 µL 0.5X TE. Libraries were prepared with the NEB Next II Ultra DNA library kit (NEB, E7645L) using 500 pg DNA input. Library quality was verified using fragment analysis (Fragment Analyzer, Advanced Analytical) and submitted for sequencing on an Illumina NextSeq 500 or 2000 instrument.