title: RNA-Seq of total mixed environmental sample: lake water
design: We sampled 21 mainland European freshwater lakes from the Sierra Nevada (Spain) to the Carpathian Mountains (Romania) and from northern Germany to the Apennines (Italy) covering a trophic gradient as well as an altitudinal range from 38 to 3110m a.s.l. All samples were taken from the edge of the water body using a telescope sampling vessel (~3m) in August 2012.  Water was filtered onto 0.2cm polycarbonate filters until the filters were blocked in order to approximate the amount of particulate matter collected per filter in the field. Once the filters fell dry after the filtration process they were immediately stabilised in RNA-stabilisation solution (LifeGuard Soil Preservation Solution, MoBio Laboratories Inc., Carlsbad, CA) and flash frozen in liquid nitrogen. The cooling chain was ensured by transportation in Cryo Shipper cooling vessels (Chart/MVE, Ball Ground, USA) until the samples reached Essen University laboratories. Afterwards they were stored at -80rC until further processing. RNA was extracted using the TRIzol method. RNA extraction was carried out under sterile conditions using TRIzol (Life Technologies, Paisley, Scotland ? protocol modified). Filters were ground in liquid nitrogen and incubated for 15min in TRIzol. Chloroform was added and the mixture was centrifuged to achieve separation of phases. The aqueous phase was transferred to a new reaction tube, and RNA was precipitated using isopropanol (incubation for 1h at -20rC and centrifugation). The RNA pellet was washed three times in 75% ethanol and resuspended in DEPC water. RNA integrity was checked by both gel electrophoresis and bioanalyser graphs and only samples which passed this test were used for subsequent sequencing. Our analyses focus on the relative contribution of different taxa and of different pathways. Thus, we normalised our sampling pipeline for approximately similar biomass/RNA rather than water volume as we did not aim at absolute levels of activity but at the relative composition of the RNA pool. Thus moderate deviations in the absolute amount of RNA do not affect the downstream analyses. Nevertheless, we ensured to process similar amounts of RNA per sample by a two-step process: First, in the field each filter was loaded with approximately similar amounts of particulate matter (the filtration pressure was used as a field proxy) and, second, the number of filters pooled for subsequent analyses were adjusted depending on the amount of RNA extracted per filter. This pooling further counter-balanced a potential micro-patchiness of the plankton community. The analysis thus was based on the RNA originating from approximately 1-5 billion (1-5 * 109) individuals including several million individual eukaryotes, corresponding to several hundred (highly eutrophic lakes) to several liters (oligotrophic lakes) of water. Preparation of cDNA libraries as well as sequencing was carried out using an Illumina HiSeq platform (Eurofins MWG GmbH, Ebersberg, Germany). Two amplified, short-insert cDNA libraries (poly-A-enriched mRNA and total RNA) with an insert size of 150-400 bp were prepared per sample, individually indexed for sequencing on HiSeq 2000, sequenced using the paired-end module (v3 chemistry) and finally demultiplexed. Libraries based on total RNA comprise predominantly rRNA fragments and therefore provide insights into the composition of the active fraction of the community whereas poly-A-enriched libraries focus on eukaryotic nucleus encoded functional genes and thereby provide deeper insights into the eukaryotic functional diversity.