Casava software 1

Template molecules for high throughput DNA sequencing were prepared from total RNA using an mRNA-Seq Sample Preparation Kit (Illumina, San Diego, CA, USA) following the manufacturer’s protocol. The library was quantified using an Agilent 2100 bioanalyzer. Each library (8 pM) was subjected to cluster amplification on a Single Read Flow Cell v4 using a cluster generation instrument (Illumina). Sequencing was performed on a Genome Analyzer GAIIx with 37 cycles, using Cycle Sequencing v4 reagents (Illumina). Image analysis and base calling were performed using Real Time Analysis version 1.13 (Illumina). Sequence libraries for each sample were processed using CASAVA Software 1.8.2 (Illumina) to produce 35-bp sequence data in fastq format. Fastq data were previously deposited in the DNA Data Bank of Japan (DDBJ) under accession number DRA005850. The datasets generated and/or analyzed during the current study are available in the DDBJ repository, accession number DRA005850.

RNA prepared from biopsies obtained at diagnosis (Day 0) and on the fourth day of neoadjuvant treatment for enrolled dogs (Day 4) was quantified and assessed for quality as described
^{11 (link),
22 (link)}. Briefly, total RNA was quantified using a fluorimetric RiboGreen assay and the total RNA integrity was assessed using capillary electrophoresis in the Agilent BioAnalyzer 2100 to generate RNA Integrity Numbers (RIN). Samples passed a QC step if they contained >1 µg with a RIN >8. Next-generation RNA sequencing (RNAseq, 50-bp paired-end) with HiSeq 2000 (Illumina) was done at the University of Minnesota Genomics Center (UMGC) in 14-paired (pre- and post-treatment) samples and two additional pre-treatment samples as described
^{22 (link)}. A minimum of ten million read-pairs was generated for each sample. Illumina’s CASAVA software 1.8.2 was used for verifying the quality of the sequence data and for FASTQ file generation and de-multiplexing. FASTQ files and processed data files are available through the National Center for Bioinformatics (GSE93516).