Nebnext multiplex

Using the NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (NEB #E7370S/L), DNA sequencing libraries were prepared for the mononucleosomal-sized and subnucleosomal-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 sample, then held at 30 °C for 30 min and then at 65 °C for 30 min. 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 °C for 15 min. 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 μl 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 and 400 bp and there were no adaptor or primer dimers.