Directional RNA-Seq for MHV68 Transcriptome

Total RNA was isolated from TRIzol (Life Technologies) as described previously (6 (link)) at 6 or 18 hpi. Poly(A)-selected RNA was chemically fragmented using RNA fragmentation reagents (Life Technologies) and purified using RNeasy MinElute cleanup columns (Qiagen). Directional RNA-Seq libraries were generated using 600 ng of fragmented 18-hpi RNA according to Illumina’s directional mRNA-Seq protocol or 200 ng of 6 hpi RNA according to Illumina’s directional Tru-Seq protocol. Nucleotides with a Phred quality score less than 20 were trimmed from the 3′ ends of raw Illumina reads. Trimmed reads with a mean quality score less than 10 or a length less than 20 were discarded. Reads were then mapped to mouse rRNA, MHV68 genome (GenBank accession number U97553), and mouse genome sequences (Build 37) with the short-read aligning program Bowtie-0.12.5 (60 (link)). Bowtie alignments were performed using the parameter setting “-best” and the settings “-e 420” and “-e 600” for 76-nucleotide and 100-nucleotide reads, respectively. Bowtie output from the MHV68 genome mapping was converted to WIG (wiggle track format) files of read depth coverage, on a log₂ scale and visualized using Gbrowse (http://www.gbrowse.org). Correlation coefficients between read depth coverage, and tiled array signals were calculated using Spearman’s ranked correlation coefficient in the R statistical environment.