Uninjured adult planarians of the indicated species were pooled and homogenized in 600 ml of TRIzol Reagent (Ambion/Life Technologies). RNA isolation for each species was performed as indicated in the reagent manual. Briefly, phase separation using chloroform was performed, followed by RNA precipitation using isopropanol. The RNA pellets were washed with 75% ethanol and resuspended in 100 ml nuclease-free water to an approximate concentration of 550 ng/ml (P. gracilis), 491 ng/ml (Girardia sp.), 1566 ng/ml (S. mediterranea, asexual), and 764 ng/ml (S. mediterranea, sexual). mRNAseq libraries were generat... [more]

To create a set of non-redundant likely protein coding sequences, all sequences were translated into longest open-reading frames using the software package Transdecoder (Version 2.01). Sequences with open-reading frames of at least 300 nucleotides (100 amino acids) were collected and oriented in the direction of the open-reading frame. For each species these oriented sequences were clustered cd-hit software package (version 4.6) with the longest sequence from each cluster being chosen as the representative sequence.