Nebnext ultra directional library kit

RNA was isolated as described previously from two replicates of S. venezuelae explorer cells growing beside S. cerevisiae for 14 days, and two replicates of S. venezuelae alone grown for 24 hr on YPD agar plates (we were unable to isolate high quality RNA from S. venezuelae alone at later time points). For all four replicates, ribosomal RNA (rRNA) was depleted using a Ribo-zero rRNA depletion kit. cDNA and Illumina library preparation were performed using a NEBnext Ultra Directional Library Kit, followed by sequencing using unpaired-end 80 base-pair reads using the HiSeq platform. Reads were aligned to the S. venezuelae genome using Bowtie 2 (Langmead and Salzberg, 2012 (link)), then sorted, indexed, and converted to BAM format using SAMtools (Li et al., 2009 (link)). BAM files were visualized using Integrated Genomics Viewer (Robinson, 2011 (link)), and normalization of transcript levels and analyses of differential transcript levels were conducted using Rockhopper (McClure et al., 2013 (link)). RNA-seq data has been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE86378 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=idmrgcmexranpun&acc=GSE86378).

Total RNA was harvested as described above. RNA quality was assessed on an Agilent Tapestation Bioanalyzer. All samples showed an RNA Integrity Number (RIN) greater than 7. An mRNA sequencing library was prepared with the NEBNextUltra directional library kit and the TruSeq stranded mRNA kit (Illumina). Paired end sequencing (2x75cycles) was performed on an Illumina NextSeq 500 obtaining over 25 million reads per sample. Reads were aligned to the HPV16 (NC_001526.3) genome using STAR_2.4.2a and counted using RSEM 1.2.31.