2.4 | Preparation steps, RNA extraction and
RNA-sequencing
In order to successfully extract RNA, the samples were lyophilized at
-58 °C and 31 µbar for 12 hours with a VirTis Sentry 2.0 (SP Scientific)
to render the (green algal) cell walls brittle. Afterwards, the samples
were again frozen in liquid nitrogen and pulverized with a bead mill
(TissueLyser II, Qiagen). RNA was isolated with the innuPREP Plant RNA
Kit (Analytik Jena) according to the manufacturer’s instructions using
the PL lysis buffer which resulted in the best RNA recovery in initial
tests. RNA quality was assessed on a 2100 Bioanalyzer Instrument with
the RNA 6000 Nano assay (Agilent). RNA libraries were constructed with
dual indexing using the TruSeq® Stranded mRNA Library Prep kit
(Illumina), which included a poly-A selection step to only sequence
undamaged eukaryotic mRNA and exclude ribosomal RNAs. Initially,
libraries with a mean fragment length of 300 bp were sequenced on a
MiSeq platform (Illumina) with the MiSeq Reagent Kit v3 (2x75bp) at the
University of Iceland in order to generate a reference transcriptome.
Additionally, they were sequenced on the HiSeq 3000/4000 SR platform
(Illumina), producing single-end reads of 50 bp at the Biomedical
Sequencing Facility in Vienna which were used for gene expression
analyses.