GSM4717241: anoxic_5; Laxus oneistus; RNA-Seq
RNA was extracted using the NucleoSpin RNA XS Kit (Macherey-Nagel). Briefly, sets of 50 worms in RNA storage solution were thawed and the worms were transferred into 90 µl lysis buffer RA1 containing Tris (2-carboxyethyl) phosphine (TCEP) according to the manufacturer. The remaining RNA storage solution was centrifuged to collect any detached bacterial cells (10 min, 4°C, 16,100 x g), pellets were resuspended in 10 µl lysis buffer RA1 (plus TCEP) and then added to the worms in lysis buffer. To further disrupt cells, suspensions were vortexed for two minutes followed by three cycles of freeze (-80°C) and thaw (37°C), and homogenization using a pellet pestle (Sigma-Aldrich) for 60 s with a 15 s break after 30 s. Any remaining biological material on the pestle tips was collected by rinsing the tip with 100 µl lysis buffer RA1 (plus TCEP). Lysates were applied to NucleoSpin Filters and samples were processed as suggested by the manufacturer, including an on-filter DNA digest. RNA was eluted in 20 µl RNase-free water. To remove any residual DNA, a second DNase treatment was performed using the Turbo DNA-free Kit (Thermo Fisher Scientific), RNA was then dissolved in 17 µl RNase-free water, and the RNA quality was assessed using a Bioanalyzer (Agilent). To check whether all DNA was digested, real-time quantitative PCR using the GoTaq qPCR Master Mix (Promega) was performed targeting a 158 bp stretch of the sodB gene using primers specific for the symbiont (sodB-F: GTGAAGGGTAAGGACGGTTC, sodB-R: AATCCCAGTTGACGATCTCC, 10 µM per primer). Different concentrations of genomic Ca. T. oneisti DNA were used as positive controls. The program was as follows: 1x 95°C for 2 min, 40x 95°C for 15 s and 60°C for 1 min, 1x 95°C for 15 s, 55°C to 95°C over 20 min. Next, bacterial and eukaryotic rRNA was removed using the Ribo-Zero Gold rRNA Removal Kit (Epidemiology) (Illumina) following the manufacturer's instructions, but volumes were adjusted for low input RNA (Alberti et al., 2014). In short, 125 µl of Magnetic Beads Solution, 32.5 µl Magnetic Bead Resuspension Solution, 2 µl Ribo-Zero Reaction Buffer and 4 µl Ribo-Zero Removal Solution were used per sample. RNA was cleaned up via ethanol precipitation, dissolved in 9 µl RNase-free water, and rRNA removal was evaluated using the Bioanalyzer RNA Pico Kit (Agilent). Strand-specific, indexed cDNA libraries were prepared using the SMARTer Stranded RNA-Seq Kit (Takara Bio USA). Library preparation was performed according to the instructions, with 8 µl of RNA per sample as input, 3 min fragmentation time, two rounds of AMPure XP Beads (Beckman Coulter) clean-up before amplification, and 18 PCR cycles for library amplification. The quality of the libraries was assessed via the Bioanalyzer DNA High Sensitivity Kit (Agilent). Libraries were sequenced on an Illumina HiSeq 200 instrument (single-read, 100 nt) at the next generation sequencing facility of the Vienna BioCenter Core Facilities (VBCF, https://www.viennabiocenter.org/facilities/). Demultiplexing was provided by the VBCF.
GEO
GSM4717241