GSM1893468: wt replicate 2; Saccharomyces cerevisiae; MNase-Seq
Thawed cell pellets were resuspended in 600 μL of 50 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, Roche protease inhibitor cocktail (PIC) and 0.2 mM PMSF and lysed by bead beating. The resulting lysate was centrifuged at 15,000g for 30 minutes and the chromatin pellet was washed and resuspended in 900 μL NP-S buffer (0.5 mM sperimidine, 1 mM β-ME, 0.075% NP-40, 50 mM NaCl, 10 mM Tris-Cl pH = 7.4, 5 mM MgCl2, 1 mM CaCl2, EDTA-free PIC, 2mM PMSF). The resuspended pellet was added to 100μL NP-S buffer containing 200 units micrococcal nuclease (MNase) and incubated at 37°C for 1 hour. The reaction was stopped by addition of EDTA to 10 mM. Crosslinks were reversed by the addition of 1% SDS and 40 μg proteinase K followed by incubation at 65 °C overnight. DNA was purified by PCI extraction and RNase A treated. Library construction for the Illumina platform was performed using a custom procedure for paired-end sequencing. Briefly, 2–10 ng of ChIP material was end-repaired and A-tailed before being ligated to TruSeq PE adaptors. In between each reaction, the material was purified using phenol:chloroform:isoamyl alcohol extraction followed by ethanol precipitation. The resulting material was then amplified in the Phusion HF master mix (NEB) using TruSeq PE PCR primer 1.0 and custom indexed multiplexing primers [5′ AAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC 3′, where “NNNNNN” corresponds to unique hexamer barcodes]. PCR amplification was performed as follows: denaturation at 98 °C for 60 s; eight cycles of (98 °C, 30 s; 65 °C, 30 s; 72 °C, 30 s), and a final extension at 72 °C for 5 min. Amplified libraries were purified using 0.8 (vol) Agencourt AMPure XP solid phase reversible immobilization paramagnetic beads and eluted in 10 mM Tris⋅HCl pH 8.5. An aliquot of each library was run on an Agilent High Sensitivity chip to check the size distribution and molarity of the PCR products.    Equimolar amounts of indexed, amplified libraries were pooled, and fragments in the 200–600 bp size range were selected on an 8% (wt/vol) Novex TBE PAGE gel (Invitrogen). An aliquot (1 μL) of the library pool was run on an Agilent High Sensitivity chip to confirm proper size selection and measure DNA concentration. The pooled libraries were diluted to 15 nM and their concentration was confirmed using the Quant-iT dsDNA HS assay kit and Qubit fluorometer (Invitrogen).