Nucleosome Mapping by MNase-seq

The nucleosome library (0.2 pmol/µl) is digested by MNase (0.05 U/µl) in nuclease digestion buffer (10 mM Tris–HCl, pH 8.0, 2 mM CaCl₂) for a time course of 0 (no MNase used), 5 and 10 min at 37°C. After the defined incubation time, digestion was stopped (2% SDS, 40 mM EDTA). Proteinase K (16 µg) is added to each sample, and the reaction is incubated at 55°C for 1 h. The DNA is purified from the reaction and concentrated using a Qiagen MiniElute Purification Kit. The DNA concentration of each sample is determined by the Invitrogen Quant-iT dsDNA Assay Kit and equalized. Illumina sequencing libraries were generated using NEBNext Ultra II DNA library prep kit. Individual samples are multiplexed and sequenced on an Illumina MiSeq 2 × 150. MNase-seq sequencing results are quality filtered (q > 30) and adapter trimmed using Cutadapt (26 ). The quality reads are merged and mapped to the 7500 nucleosome library sequences using Vsearch (27 (link)). The read counts and end positions are used to determine MNase protection, which is a measurement of the percentage of reads for a specific nucleosome base pair location. MNase protection is calculated for each base pair as the ratio of base pair coverage/total reads for that specific nucleosome.