title: GSM2877720: wt_YPD_48h_H3_1_no-spike-in; Saccharomyces cerevisiae; ChIP-Seq
library_construction_protocol: Percoll gradients (1 for 7.5 hr or 2 for 24/48 hr timepoints) were formed by vortexing 1.16ml Percoll (Sigma P1644) with 42µl 5M NaCl, 98 µl water in 2ml tubes and centrifuging 15 min at 15,000 g, 4 °C. Cells were defrosted on ice, re-suspended in ~250 µl cold PBSE per gradient and layered on the pre-formed gradients. Gradients were centrifuged for 20 min at 1,000 g, then the upper phase and brown layer of cell debris removed and discarded. 1 ml PBSE was added, mixed by inversion and centrifuged 1 min at 2,000 g to pellet the cells, which were then re-suspended in 1ml PBSE per timepoint (re-uniting samples split across two gradients). 25 µl Streptavidin magnetic beads were added (Miltenyi Biotech 1010007) and cells incubated for 30 min on a wheel at room temperature. Meanwhile, 1 LS column per sample (Miltenyi Biotech 1050236) was equilibrated with cold PBSE in 4 °C room. Cells were loaded on columns and allowed to flow through under gravity, washed with 8 ml cold PBSE and eluted with 1ml PBSE using plunger. Cells were re-loaded on the same columns after re-equilibration with ~500 µl PBSE, washed and re-eluted, and this process repeated for a total of three successive purifications. Final elution was to 1.5 ml tubes (Treff 1130153). 50 µl cells were set aside for quality control, while the remainder were pelleted by centrifugation and processed directly for ChIP.  Cells were re-suspended in 50 μl cold CLB (50mM HEPES pH 7.0, 14mM NaCl, 1mM EDTA, 1% Triton X100, 0.1% sodium deoxycholate, 0.1% SDS, cOmplete Protease Inhibitors (Roche 1836170)) with 50 µl zirconium beads and lysed with 5 cycles of 30 s 6500 ms-1 / 30 s ice in an MP Fastprep bead beater then decanted to a Covaris 520045 microTUBE and topped up to 130 µl with CLB. Sonication was performed on a Covaris 220: Duty Factor 5 %, PIP 130 W, 900s, 200 cycles per burst, 8.9 ºC. Samples were decanted to a 1.5 ml tube and Covaris tube washed with CLB and combined to give 180 μl total volume, which was centrifuged 5 min top speed at 4 ºC and the pellet discarded. Lysate was pre-cleared by incubation with 10μl of pre-equilibrated Protein-G magnetic beads (Life Technologies 10004D), then 30 μl set aside for total DNA extraction and 50 μl used per immunoprecipitation (IP). Antibodies were added at 1:50 dilution (rabbit anti-H3, CST 2650 or rabbit anti-H3K4me3, CST 9751), one IP was processed without antibody, and IPs were incubated over night at 4 ºC on a wheel.   10μl pre-equilibrated protein-G beads in 25μl CLB were added to each IP and incubated 2-3 hrs at 4 ºC on a wheel. Beads were then washed once each at room temperature for 5 minutes on a wheel with: CLB, CLB with 0,5 M NaCl, wash buffer (10 mM Tris pH 8.0, 0.25 M LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA), TE, then eluted overnight at 65 ºC with 200 μl elution buffer (50 mM Tris/HCl pH 8.0, 10 mM EDTA, 1% SDS). Input samples were diluted to 200 μl with TE, treated with 1 μl 1 mg/ml RNase A at 37 ºC for 1 hour then incubated overnight at 65 ºC. Input and IP samples were treated with 1 μl proteinase K for 2 hours at 55 ºC, then purified by phenol:chloroform extraction, precipitated and re-suspended in 5 μl TE. libraries were synthesised using a NEBNext DNA Ultra II kit (NEB E7645) with modifications: Concentration of DNA was increased by reducing the volumes of the end repair and ligation steps 5-fold, with reagent quantities reduced in kind. After clean-up with 0.9x AMPure beads, PCR in 50 μl volumes according to manufacturer's instructions. Libraries were purified with 0.9x AMPure beads and eluted in 30μl, then a size selection was performed by incubating first with 18μl AMPure beads, discarding the beads then purifying the remaining DNA from the supernatant with an additional 9 μl AMPure beads.