title: Sample 13
organism: Heliconius melpomene
description: Protocols: Pupae of Heliconius melpomene were obtained from the Costa Rica Entomological Supply, Costa Rica. They were then frozen at -80C 1-3 days post-eclosure, and RNA was extracted. RNA was extracted from the head, antennae, legs and mouthparts (labial palps + proboscis) from individual butterflies using TRIzol (Life Technologies, Grand Island, NY) and purified using a NucleoSpin RNA II kit (Macherey-Nagel, Bethlehem, PA). Purified RNA was quantified using a Qubit 2.0 Fluorometer (Life Technologies, Grand Island, NY) and quality checked using an Agilent Bioanalyzer 2100. A TruSeq RNA Sample Preparation Kit v2 (Illumina, San Diego, CA) was used to prepare sequencing libraries. Libraries with distinct adapter sequences were quantified, quality checked, normalized, and pooled according to thier concentrations. Pooled libraries were run on a 2% agarose gel. A Geneclean III kit (MP Biomedical, Santa Ana, CA) was used to recover DNA from the gel (240-600 bp), and Agencourt AMPure XP (Beckman Coulter, Brea, CA) beads were used for a second purification.