Snails for transcriptome analyses were originally collected from different sites in France (MOTU3), Switzerland (MOTU5) and Germany (R. auricularia) between 2005 and 2011 (Additional file 6 ) and kept in water basins untill the start of this project. Information on treatment and sequence generation for R. balthica snails is reported in a previous study and documented in Feldmeyer et al. [30 (link)]. We placed individual snails in different temperature treatments. In 2011 snails were transferred to 500 ml glass jars and underwent one of six treatments, with two individuals per population, per treatment: Three days at 10 °C or 30 °C with and without aeration, 10 min temperature shock at either 4 °C or 36 °C before storage in RNAlater (Qiagen). RNA was isolated following the RNeasy maxi kit protocol (Qiagen). cDNA production, normalization, and sequencing was performed by GenXPro GmbH (Frankfurt, Germany). Due to technical issues and different time points of sequencing, the three libraries were sequenced on different platforms, MOTU3 + R. auricularia on the 454 FLX system, and MOTU5 on an Illumina HiSeq 2000. Since we were interested in sequence divergence between species, the usage of different platforms will not impair data analyses as might be expected for gene expression studies.
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