Micrococcal Nuclease-seq

Micrococcal nuclease (MNase)-seq was performed using the EZ Nucleosomal DNA Prep Kit (Zymo Research #D5220) following the manufacturer’s protocol. Briefly, nuclei isolated from 1 million iciHHV-6A and 293-HHV-6A cells were treated with varying concentrations of MNase (final concentrations of 0.07, 0.1, 0.2, 0.3 U/100 μl reaction) and incubated at room temperature for 5 min. The reaction was terminated with MNase stop buffer, and following nucleosomal DNA purification, libraries for each individual titration were prepared with the NEB Ultra II Library Prep Kit (#E7645S) following the manufacturer’s protocol. Libraries were purified using 0.8X AMPure beads and quantified using Qubit (Life Technologies). Library quality and presence of mono-, di-, and tri-nucloesomes were observed using the Agilent 2,100 Bioanalyzer High-Sensitivity DNA kit. Barcoded libraries were pooled and sequenced on an Illumina HiSeq 2,500 instrument lane at the Genomics Core Facility (University of Texas Health Science Center, San Antonio, TX) to obtain 80-bp paired-end reads. Sequences were aligned to the human (hg38), HHV-6A ({"type":"entrez-nucleotide","attrs":{"text":"NC_001664.2","term_id":"224020395","term_text":"NC_001664.2"}}NC_001664.2), and custom HHV-6A-GFP genome using Bowtie2 (Langmead and Salzberg, 2012), and the bamCoverage command in deepTools was used to filter out reads with insert sizes <50 bp and >500 bp and to generate bigwigs (Ramírez et al., 2016). Profiles were generated for individual titration points, and as it has been previously reported that nucleosome occupancy can change across the genome in response to MNase treatment (Maehara and Ohkawa, 2016; Mieczkowski et al., 2016; Mueller et al., 2017), data for each titration were merged based on the cell line. Data were processed using DANPOS as described previously (Chen et al., 2013).