One microgram of high quality RNA from each sample was used for sequencing. Stranded RNA-seq libraries were prepared using TruSeq SBS Sequencing kit version 3 (Illumina, San Diego, CA). Libraries were barcoded and pooled and sequenced on two lanes on a HiSeq2500. Reads were single-end, stranded, and 100 nt in length. Sequencing results were processed by CASAVA 1.8 (Illumina, San Diego, CA).
Data quality, including base quality per position across reads, GC content, and distribution of sequence length, was initially assessed with FastQC (Andrews 2010 ). Reads were processed with flexbar (Dodt et al. 2012 (link)) in two passes: the first to trim adapters, remove low quality reads, and remove reads <35 bp in length, and the second to remove polyA tails. Subsequently, reads that mapped to fox mitochondrial DNA sequences from NCBI (accession numbers JN711443.1, GQ374180.1, NC_008434.1, and AM181037.1) using Bowtie2 (Langmead and Salzberg 2012 (link)) were discarded. Similarly, any remaining reads that mapped to ribosomal DNA sequences were discarded.
Data quality, including base quality per position across reads, GC content, and distribution of sequence length, was initially assessed with FastQC (Andrews 2010 ). Reads were processed with flexbar (Dodt et al. 2012 (link)) in two passes: the first to trim adapters, remove low quality reads, and remove reads <35 bp in length, and the second to remove polyA tails. Subsequently, reads that mapped to fox mitochondrial DNA sequences from NCBI (accession numbers JN711443.1, GQ374180.1, NC_008434.1, and AM181037.1) using Bowtie2 (Langmead and Salzberg 2012 (link)) were discarded. Similarly, any remaining reads that mapped to ribosomal DNA sequences were discarded.
