title: GSM6124912: histone H4 ChIP-seq, ChIP DNA: Spn1-AID + DMSO 6; Saccharomyces cerevisiae; ChIP-Seq
library_construction_protocol: 150 mL of each culture was transferred to a flask that was at room temperature, and 36 mL of 4°C YPD was added to each. These cultures were then fixed with 1% formaldehyde for 30 minutes while shaking at room temperature. The crosslinking was quenched by adding glycine to each culture to a final concentration of 125 mM and by shaking for 10 minutes at room temperature. The cells were pelleted, washed twice with cold 1x TBS (100 mM Tris, 150 mM NaCl pH 7.5), and flash frozen. FLR196 and FLR142 cell pellets were resuspended in 800 μL of LB140 (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1%. Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 100 μg of leupeptin, 100 μg of pepstatin A, 8.71 mg of PMSF, 3.08 mg of 1M DTT), and lysed by bead-beating at 4°Cfor 8 minutes with 4-minute incubations on ice after every minute of bead-beating. The FWP10 pellets were treated similarly, but they were lysed by bead-beating at 4°C for 11 minutes. The lysate was collected by centrifuging the samples at 14,000 rpm for 5 minutes at 4°C. After  discarding the supernatant, the pellet was washed with LB140. The final pellet was washed in 580 μL of LB140 and sonicated in a QSonica Q800R machine for 10 minutes (30 seconds on, 30 seconds off, 70% amplitude) to obtain fragments of about 100-500 bp. The protein concentrations of all samples were measured by Bradford (Bradford 1976). To prepare the IP reactions, about 250 μg of S. cerevisiaechromatin was used as starting material, and we added 10% the mass of chromatin as spike-in from S. pombe to each sample. We added to each sample 250 μL of WB140 (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate), and the volume was brought up to 500 μL with LB140. Then, 40 μL of each sample were set aside as input for later processing at the reverse crosslinking step, described later. Two sets of IPs were prepared per sample with the following antisera: 3 μL of anti-H4 (AV94), kindly provided by Dr. Alain Verreault, and 16.7 μL of anti-H4ac (Millipore Sigma, 06-866, which targets histone H4ac on lysines 5, 8, 12, and 16), volumes that were optimized by ChIP-qPCR. The immunoprecipitation reactions were incubated at 4°C with end-over-end rotation overnight (∼14-16 hours). Aliquots of Dynabeads Protein G for Immunoprecipitation (Invitrogen) and Dynabeads Protein A for Immunoprecipitation (Invitrogen) were transferred to Eppendorf tubes, and the buffer was removed. A volume of WB140 equivalent to the initial volume of beads was used to wash the beads twice with WB140, and the beads were kept in the second wash to make a slurry. Fifty μL of the Protein G slurry were added to each of the H4 IPs, 278 μL of the Protein A slurry were added to the H4ac IPs, and the reactions were incubated at 4°C with end-over-end rotation for four hours. The beads were washed twice (first wash for one minute and second wash for 5 minutes with nutation) with WB140, twice with WB500 (50 mM HEPES-KOH pH 7.5, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate), twice with WBLiCl (10 mM Tis-HCl pH 7.5, 250 mM LiCl, 1 mM EDTA, 0.5% NP-40, 0.5% sodium deoxycholate), and once with TE (10 mM Tris-HCl pH 7.4, 1 mM EDTA) for 5 minutes. The IP material was eluted from the beads twice with 100 μL of TES (50 mM Tris-HCl pH 7.4, 10mM EDTA, 1% SDS) at 65°C for 30 minutes first and then for 15 minutes. The saved inputs were thawed, and 60 μL of LB140 and 100 μL of TES were added. To reverse crosslinking, the eluates and the inputs were incubated at 65°C overnight. To degrade RNA, 2 μL of RNAse A/T1 (Thermo Fisher, EN0551) were added to each sample, and the samples were incubated at 37°C for one hour. To degrade protein, a mix of 7 μL of proteinase K (Roche), 1 μL of glycogen (Sigma G1767-1VL, 20 mg/mL), and 250 μL of TE was added to each sample, and the samples were incubated at 50°C for two hours. The DCC-5 kit (Zymo Research, D4014) was used to purify DNA (20-μL elution). The libraries were built with 10.25 μL of DNA as starting material and using the GeneRead DNA Library I Core Kit (Qiagen, half of the volumes suggested by the manufacturer instructions were used) and custom barcodes (Wong, Jin, and Moqtaderi 2013). The samples were purified twice using 0.7x volume SPRI beads (A63881). PCRs were performed to determine number of amplification cycles, which ranged from 11 to 19, and to perform final library amplifications. Double-sided selection was performed to purify the samples a second time using 0.56x volume SPRI beads first and 0.7x volume SPRI beads second. The size distribution, quality, and concentration of the samples was assessed by running them in an Agilent Bioanalyzer.