RNA-seq of white Heliconius cydno galanthus: male 96-108 h pupa central brain
We dissected individuals in cold PBS and immediately placed dissected tissues into RNAlater (Ambion, USA). Tissues were stored in RNAlater at -80oC until RNA extractionusing TRIzol (Ambion, USA). High quality (RIN > 7) RNA samples were treated with Turbo DNAse (Invitrogen, USA), then 1 ug was used as input for poly-A selection and RNA-seq library preparation using the NEBNext Poly(A) mRNA Magnetic Isolation Module and NEBNext UltraII Directional RNA Prep Kit following the manufacturers instructions with minor modifications. RNA fragmentation was performed for 10 min at 94oC. We used theNEBNext Multiplex Oligos for Illumina dual-index adapters. Double-sided selection was performed after adapter ligation to enrich for ~300 bp - 500 bp fragments. Final libraries were PCR amplified for 11 cycles. RNA-seq libraries were pooled and sequenced 2x100 bp on a NovaSeq 6000 at the University of Chicago Functional Genomics Facility.