GSM6049375: Non-stressed_C8_Rep1; freshwater metagenome; RNA-Seq
Samples were grinded into liquid nitrogen. Afterwards the RNeasy Plant Mini kit (QIAGEN, Inc., Valencia, CA) was used according to the manufacturer. RNA was eluted two times (30µL and 10µL) with 60°C elution buffer. Then, DNA contaminations were digested using the Ambion Turbo DNA-free kit (Thermo Fisher Scientific, Inc., Waltham, MA) following the instructions. To remove DNase buffer the RNA was precipitated by ethanol. The final dried pellet was dissolved into 20µL of RNase-free water and incubated for 8 minutes at 58°C. After cooling-down on ice, and a brief centrifugation, the RNA samples were stored at -80°C. 100 ng total RNA was used for rRNA depletion. Ribosomal RNAs were removed from total RNA using the Ribo-Zero Magnetic Kit (Illumina, San Diego, CA, USA) according to the manufacturer`s low input instructions. To improve rRNA depletion, rRNA probes from Ribo-Zero bacteria kit (MRZMB126) and plant kit (MRZPL1224) were combined. A strand-specific library for transcriptome sequencing was prepared using the ScripSeqv2 Kit (Illumina) following the manufacturer`s instructions. The library was checked by using the Agilent 2100 Bioanalyzer system with a High Sensitivity DNA Kit (Agilent) according to the manufacturer`s instructions. Library concentration was determined by using the Qubit 2.0 instrument using the Quant-iT dsDNA High Sensitivity kit (Thermo Fisher Scientific) according to the manufacturer`s instructions. Single libraries were normalized by equimolar pooling. The library pool was size-selected in a range of 150-600 bp using a preparative agarose gel in combination with MinElute gel extraction kit (Qiagen) according to the manufacturer's protocol. 12 pmol of the library pool were clustered on 7 lanes of an Illumina paired-end flow cell and 2x100 bp were sequenced according to the manufacturer's instructions using the v3 Cluster Generation and SBS Sequencing Kits (Illumina, San Diego, CA, USA) on a HiSeq2000 system.