Instrument: Illumina HiSeq 2000
Strategy: MNase-Seq
Source: GENOMIC
Selection: MNase
Layout: SINGLE
Construction protocol: Nucleosome mapping has been performed according to Rando O. (Rando, 2010) with minor modifications. Stationary phase yeast culture (≈6 OD/ml) is cross-linked with 1% formaldehyde for 20 minutes with occasional rotation at room temperature. The reaction is quenched with glycine for 5 min at room temperature. About 60 OD of yeast cells are washed twice with PBS buffer and then resuspended in 2 ml of Z buffer (1M sorbitol, 50 mM Tris-HCl pH 7.4 with freshly added 10 mM β-mercaptoethanol) containing 3.6 mg of Zymolyase-20T (from Arthrobacter luteus, AMS Biotechnology) and incubated at 37°C in agitation. After 45 min the same amount of Zymolyase is added and each sample which is incubated for an additional 45 min at 37°C in agitation. Spheroplasts are then pelleted by centrifugation for 5 min at 4°C at 1,500 g. The pellet is washed once with NP-buffer, then resuspended in 1.6 ml of NP-buffer and divided in 3 tubes. An increasing amount of MNase (Sigma, N3755) is added to each tube: 0.25 U, 0.5 U and 1 U. After incubation for 20 min at 37°C, each reaction is stopped by the addition of SDS and EDTA to a final concentration of 1% and 10 mM, respectively. The reaction is then treated with 0.2 mg/mg of Proteinase K (NEB) at 65°C overnight. The sample is then purified with 2 rounds of phenol:chloroform (1:1) and the aqueous solution precipitated. The resuspended DNA pellet is treated for 1 h with RNase A at 37°C. For the naked DNA digestion, 200 ng of extracted DNA are incubated at 37°C with 0.01 U of MNase. After 7 minutes the reaction is stopped as described before. Both naked and RNaseA-treated nucleosomal DNAs are then purified using 1.8 volumes of AMPure XP beads Libraries were prepared using NEBNext DNA Library Prep Master Mix Set for Illumina (NEB, E6040S) with few modifications of the manufacturer’s protocol. Only the digestion pattern obtained with 0.5 U of MNase was used for the library preparation. The DNA is end-repaired (in half of the suggested volume), dA-tailed and 1 μl of Illumina TruSeq Adapters is added to a 40 μl ligation reaction. Purification after each step are performed using AMPure XP beads according to the protocol. The size selection step is carried out with 0.8x of AMPure beads in the first step and 0.2x of AMPure XP beads in the second step. Half of the DNA is amplified using Illumina PCR Master Mix and Illumina TruSeq PCR Primer Cocktail (TruSeq DNA Sample Prep kit v2) with the following protocol: initial denaturation at 98°C for 30 seconds; 12 cycles of 98°C for 15 seconds, 60°C for 30 seconds, 72°C for 30 seconds; final extension at 72°C for 5 minutes. The final product is purified using AMPure XP beads before being submitted for sequencing.