RNA-seq of Scyliorhinus canicula: St31 habenulae
We prepared RNA-seq libraries using the TruSeq Stranded mRNA Sample Prep Kit (20040532, Illumina, San Diego, CA, USA) according to the manufacturers instructions. Briefly, polyadenylated RNAs were selected using oligo-dT magnetic beads. The polyA+ RNAs were fragmented using divalent cations at elevated temperature and reverse transcribed using random hexamers, Super Script II (18064014, Thermo Fisher Scientific, Carlsbad, CA, USA), and actinomycin D. Deoxy-TTP was replaced by dUTP during the second strand synthesis to prevent its amplification by PCR. Double-stranded cDNAs were adenylated at their 3' ends and ligated to Illumina adapters containing unique dual indexes (UDI). Ligated cDNAs were PCR amplified in 15 cycles, and the PCR products were purified using AMPure XP Beads (A63880, Beckman Coulter Genomics, Brea, CA, USA). The size distribution of the resulting libraries was monitored using a Fragment Analyzer (Agilent Technologies, Santa Clara, CA, USA), and the libraries were quantified using the KAPA Library Quantification Kit (Roche, Basel, Switzerland). The libraries were denatured with NaOH, neutralized with Tris-HCl, and diluted to 7.5 pM.