Mitochondrial transcriptome sequencing of Physarum polycephalum
We used the Illumina protocol "Directional mRNA Library Preparation, Pre-ReleaseProtocol Rev.A.", adapting it for mtRNA sequencing.  The polyA-selection step was omitted and total mitochondrial RNA was fragmented using 15µl (~500ng) of mtRNA, 2µl Frag Buffer (Illumina) and 3 µl ddH2O. Incubation time was extended to 20min (94°C, stopped with 1 µL Stop Buffer, Illumina). An Agilent Bioanalyzer run was conducted showing the mtRNA reduced to fragments between 25 and 100bp. We decided not to implement any size selection to avoid losing naturally occuring small RNAs. The further steps of the protocol involved purification, phosphatase treatment, PNK treatment, again purification, adapter ligation, reverse transcription, amplification, and again purification using AMPure beads. The final Agilent Bioanalyzer process control run showed a library size of 168 bp on average, composed of 73 bp adapter sequences and about 95 bp insert size in which some of these inserts were composed of more than one ligated original fragment.