title: RNAseq of H. charithonia adult female legs
design: Butterflies were fresh frozen at 80C until RNA extraction. Tissue were dissected and RNA was extracted using TRIzol (Thermo Fisher Scientific, Waltham, MA) following the manufacturers instructions. RNA was purified using a NucleoSpin RNA II kit (Macherey-Nagel, Bethlehem, PA). Purified RNA was quantified using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA) and RNA integrity was checked using an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). RNA sequencing libraries were prepared using a TruSeq RNA Sample Preparation Kit v2 (Illumina, San Diego, CA). Double-stranded cDNA libraries were quantified, quality checked, normalized, and pooled according to their concentrations. Pooled libraries were run on a 2% agarose gel to size select fragments of 240600bp. DNA was recovered using a Geneclean III kit (MP Biomedical, Santa Ana, CA) and purified using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA). Sequencing was conducted at the UCI Genomics High-Throughput Facility using a HiSeq 2500 (Illumina, San Diego, CA), paired end 100-cycle sequence run.