RNA-Seq of Polynucleobacter asymbioticus with different chrysophyte community
P. asymbioticus (strain QLW-P1DMWA-1, Hahn et al. (2016b)) was co-cultured with single- and multi-species chrysophyte communities containing the species Chlorochromonas danica (strain 933-7, Pringsheim (1955)), Poterioochromonas malhamensis (strain DS, Boenigk et al. (2006) and Poteriospumella lacustris (strain JBM10, Findenig et al. (2010)) Precultures of the axenic chrysophyte species were grown at 15 C in NSY (nutrient broth, soytone, yeast extract) medium (Hahn et al., 2004). Cultures were illuminated with 75-100 E light intensity and a light:dark cycle of 16:8 h. For the experiment, cells were harvested by centrifugation at 3,000 g for 10 min at 20 C and washed in modified DY-V medium (pH 6, Andersen (2007)). P. asymbioticus was cultivated in inorganic basal medium (NSY medium supplemented with 3 g/l of each of nutrient broth, soytone and yeast extract) under continuous shaking at room temperature. Prior to the experiment, cells were harvested by centrifugation at 5,000 g for 10 min at 20 C and washed three times with DY-V (pH 6) medium. For the experiment, all samples were inoculated with the chrysophytes at the same biovolume, corresponding to approx. 4702505 m3/ml in total for each treatment and 30 million bacteria per ml, which amounts to an approx. 1000-fold higher bacterial cell number than chrysophyte cells. Experiments were run in 3 replicates for 24 h in modified DY-V (pH 6) medium under the same settings as above.  RNA extraction was carried out under sterile conditions using TRIzol (Life Technologies, Paisley, Scotland{protocol modified). Prior to the RNA extraction the stabilization solution was removed from the filters. In brief, filters were ground in liquid nitrogen and incubated for 15 min in TRIzol at 15 C. Chloroform was added, shaken vigorously and incubated for 15 min at 15 C. 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 -20 C and centrifugation). The RNA pellet was washed three times in 75% ethanol and re-suspended in diethylpyrocarbonate (DEPC) water. Sequencing was performed at the sequencing provider BGI (Hong Kong). They performed quality control of the samples, rRNA depletion, library preparation and paired-end sequencing of the RNA with a read length of 100 bp on the Illumina HiSeq 4000. Upon sequencing the data was filtered to remove adapter sequences, contaminations and low-quality reads from the raw reads.