Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Brains of wild-caught migrants and of monarchs raised indoors in LP and SP were dissected in 0.5X RNA later (Invitrogen) to prevent RNA degradation, the retinal pigmented photoreceptor layer was removed, and the brains were stored at -80oC until use. For each seasonal phenotype/photoperiodic condition, three pooled brains were collected in two replicates at ZT1, ZT4, ZT7, ZT10, ZT13, ZT16, ZT19, and ZT22. For each sample, total RNA was extracted using an RNeasy Mini kit (Qiagen). For samples from wild-caught migrants, polyA+ RNA was isolated from 2 μg of total RNA with NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs), and multiplexed libraries were prepared using the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina and NEBNext Multiplex Oligos (New England Biolabs) and amplified with 12 PCR cycles, following the manufacturer's recommendations. For samples from monarchs raised in LP and SP, multiplexed libraries were prepared by the Texas A&M AgriLife Genomics and Bioinformatics Facility using polyA+ RNA isolated from 1 μg of total RNA, and multiplexed libraries were prepared using the TruSeq Stranded mRNA Library Prep Kit (Illumina), following the manufacturer's recommendations. Libraries quality and size distribution was verified on a Bioanalyzer, libraries were quantified by real-time quantitative PCR, and 16 multiplexed libraries were mixed in equimolar ratios and sequenced on a Hi-seq 2500 (Illumina) using 50bp single end reads.