De novo assembled transcriptomes and RNA-Seq data for three species of Macrostomum flatworms. Macrostomum hystrix, Macrostomum spirale, Macrostomum pusillum.</p><p>RNA-Seq libraries for adults, hatchlings and regenerating worms, using three biological replicates per condition and species, for a total of 27 RNA pools and resulting RNA-Seq libraries. We defined adults as animals with clearly visible testes and collected 60 animals per replicate for M. hystrix and M. spirale, and 225 animals per replicate for M. pusillum. Hatchling samples consisted of a mixture of animals from various developmental stages, from freshly hatched flatworms up to early juvenile stages, but never having any visible gonads. We collected on average about 330, 650, and 1100 hatchlings for each replicate of M. hystrix, M. spirale, and M. pusillum, respectively. Due to the large number of animals needed for this experiment, hatchlings of M. pusillum and M. spirale were harvested at two time points, dissolved in Tri reagent, and stored at -80°C until RNA isolation. Animals used for the regeneration group were amputated at the level behind the ovaries and then put on ASW with diatoms and allowed to regenerate for a variable amount of time before sampling to capture animals at various stages of regeneration. For M. hystrix and M. spirale, ten animals per replicate were cut each day for six subsequent days, and on the seventh day, these animals (6x10 = 60 animals per replicate) were used for RNA isolation. M. pusillum was treated in the same way, but five times 30 animals were amputated, and total RNA was isolated on the sixth day (5x30 = 150 animals per replicate).