Transcriptome sequencing, alignment and quantification
RNA from three tissues (liver, spleen, testis) was extracted from 7 male tree sparrows from BY and 7 male tree sparrows from LJX. RNA sequencing was performed based on 150 bp paired-end reads with insert size around 350 bp using Illumina NovaSeq 6000 platform. After filtering and quality control procedures, the clean reads were mapped to reference genome using STAR v2.7.9a (Dobin et al., 2013).
The gene-level quantification approach utilizes the files of aligned reads generated during alignment and a gene transfer format (GTF) file containing gene models of tree sparrow to count the number of reads that mapped to each gene using featureCounts v2.8.1 (Liao et al., 2014). We obtained the resulting fragments per kilobase of exon per million fragments mapped (FPKM) and transcripts per million (TPM). We used the trimmed mean of the M-values (TMM) normalization to compare gene expression values among replicates from the same tissue.