Instrument: Illumina HiSeq 2500
Strategy: MNase-Seq
Source: GENOMIC
Selection: MNase
Layout: PAIRED
Construction protocol: Nuclei were isolated in nucleus isolation buffer containing: 10 mM HEPES at pH 7.8, 2 mM MgOAc2, 0.3 M sucrose, 1 mM CaCl2, and 1% Nonidet P-40. The nuclei were then pelleted by centrifugation at 1000g for 5 min at 4°C. Using the NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (NEB #E7370S/L), DNA sequencing libraries were prepared for the mononucleosomal-sized and sub-nucleosomal-sized fragments for each sample. DNA was end-prepped using NEB Prep enzyme mix, end-repair reaction buffer (10X), and 30 ng of DNA for each samples, then held at 30 degrees Celsius for 30 minutes and then at 65 degrees Celsius for 30 minutes. Adaptors were ligated onto the end-repaired samples by adding NEB Blunt/TA Ligase Master Mix, NEBNext Adaptor for Illumina, and Ligation Enhancer and incubating at 20 degrees Celsius for 15 minutes. The adaptor-ligated DNA was cleaned up using AMPure XP beads to remove any unwanted ligated products. The universal and indexed sequences were added by PCR using 23 ul of adaptor-ligated DNA fragments, NEBNext High Fidelity 2X PCR Master Mix, index primers provided in NEBNext Multiplex (NEB #E7335, #E7500) Oligos for Illumina, and Universal PCR Primers provided in NEBNext Multiplex (NEB #E7335, #E7500) Oligos for Illumina. Then PCR was done for 8 cycles (not including the initial denaturation and final extension). The adaptor-ligated DNA was cleaned up using AMPure XP beads to remove any unwanted products. The libraries were quality-checked using Agilent 2100 Bioanalyzer High-Sensitivity. Across the libraries, the samples ranged between 200-400bp and there were no adapter or primer dimers.