title: Principapillatus hitoyensis: eyes; RNA-seq
design: Total RNA was extracted using TRIZOL (Invitrogen, Carlsbad, CA) and the RNeasy MinElute Cleanup Kit (Qiagen, Hilden, Germany) according to the manufacturers protocols. Double-stranded complementary DNA was prepared following Illuminas messenger RNA (mRNA)-seq sample preparation guide (Part #1004898 Rev. D). Briefly, mRNA was isolated from total RNA using Dynal oligo(dT) beads (Invitrogen) and fragmented (~200bp) using divalent cationic ions. First-strand synthesis was performed with random hexamer primers, using Superscript II polymerase (Invitrogen). Subsequent second-strand synthesis was carried out with DNA Pol I polymerase (Invitrogen). Starting at the blunt end repair step, multiplex sequencing libraries were prepared following the multiplex protocol of Meyer and Kircher (2010), with modifications for double indexing described in Kircher et al. (2012). These libraries were sequenced according to the manufacturers instructions with 75 cycles paired end on the Genome Analyzer IIx platform (v4 sequencing chemistry and v4 cluster generation kit; Illumina, San Diego, CA).