Truseq stranded mrna sample prep kit with 96 dual indexes

Total RNA extracted from each species was sent to the National Genomics Infrastructure sequencing facility (Uppsala, Sweden). RNA libraries for sequencing were prepared using TruSeq Stranded mRNA Sample prep kit with 96 dual indexes (Illumina, CA, USA) according with manufacturer’s recommended instructions with the following changes: the protocols were automated by using an Agilent NGS workstation (Agilent. CA. USA) with purification steps65 (link)66 (link). Samples were clustered using cBot and sequenced on a HiSeq2500 (HiSeq Control Software 2.2.8/RTA 1.18.61) with a 2 × 126 setup in RapidHighOutput mode. Bcl to Fastq conversion was performed using bcl2Fastq v1.8.3 from the CASAVA software. Quality scale was Sanger/phred33/Illumina 1.8 + .
For each species two fq files were produced, one containing all left-pair reads and another containing all right-pair reads. All sequence read files were deposited in our private project account on the UPPMAX server (Uppsala, Sweden).

We used total RNA extracted from whole brain from six samples used in an earlier study quantifying differential expression in migratory and breeding willow warblers^{69 (link)} (Supplementary Table 3). The quality of the RNA was checked with a Bioanalyzer version 2100 (Agilent, CA, USA). All of the extractions had a RNA Integrity Number (RIN) of at least > 7.10. RNA libraries for sequencing were prepared using a TruSeq Stranded mRNA Sample prep kit with 96 dual indexes (Illumina) according to the instructions of the manufacturer with the exception of automating the protocols using an NGS workstation (Agilent) and using purification steps as described in Lundin et al^{70 (link)}. and Borgström et al^{71 (link)}. The raw RNA data was trimmed using cutadapt version 1.8⁷² within Trim Galore version 0.4.0 (https://github.com/FelixKrueger/TrimGalore) with default settings.
We used Stringtie version 1.3.3^{73 (link)} to create transcripts from the RNAseq data. These transcripts were not used directly in the generation of gene models, but used in the manual curation step as potential alternative transcripts. For the software, we first mapped the reads with Hisat2 version 2.1.0^{74 (link)} using default settings for stranded sequence libraries and downstream transcript analyses.

Samples of first instar larval heads, adult male antennae, and adult female antennae were prepared for RNA sequencing (see Insect Rearing and RNA Extraction; Supplementary Materials and Methods) at the National Genomics Infrastructure sequencing facility (Uppsala, Sweden). RNA libraries for sequencing were prepared using TruSeq Stranded mRNA Sample prep kit with 96 dual indexes (Illumina, CA, USA) according to the manufacturer’s instructions, with the following changes: the protocols were automated using an Agilent NGS workstation (Agilent, CA, USA) using purification steps as described45 (link)46 (link). Samples were clustered using cBot and sequenced on a HiSeq2500 (HiSeq Control Software 2.2.38/RTA 1.18.61) with a 2 × 126 setup in RapidHighOutput mode. Bcl to Fastq conversion was performed using bcl2Fastq v1.8.3 from the CASAVA software suite. The quality scale is Sanger/phred33/Illumina 1.8+.
All sequence read files were delivered to our project account on the UPPMAX Computational Science server (Uppsala, Sweden). For each sample, two fq files were produced, one containing all left-pair reads (sampleX_1.fq) and one containing all right-pair reads (sampleX_2.fq).