design: Strains were cultured in YPC medium and grown to OD650nm of 0.6 - 0.7. Total RNA was isolated using NucleoZOLTM (Machery and Nagel) and RNA samples were sent to Vertis Biotechnologie AG (Martinsried, Germany) for sequencing. Total RNA was treated with T4 Polynucleotide Kinase (NEB) and rRNA was depleted using an in-house protocol. Samples were fragmented using an ultrasonic treatment before cDNA library was prepared. For this, first-strand cDNA synthesis was performed using a 3-adapter primer and the M-MLV reverse transcriptase. After cDNA purification, the 5' Illumina TruSeq sequencing adapter was ligated to the 3' end of the antisense cDNA and the sample amplified to 10-20 ng/l using a high-fidelity DNA polymerase. Finally, cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics), samples were pooled (equimolar) and the pool size was fractionated (200  550 bp) by preparative agarose gel electrophoresis before the samples were sequenced on an Illumina NextSeq 500 system using 1x75 bp read length. Quality control was conducted on raw reads and after each processing step using fastqc (v0.11.9) (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/, accessed 09/09/20) and MultiQC (v1.10.1) (Ewels et al., 2016). Adapters were trimmed using trimgalore (v0.6.3) (https://github.com/FelixKrueger/TrimGalore, accessed  09/09/20) and reads were mapped against the H. volcanii genome with STAR (v2.7.3a) (Dobin et al., 2013). Unique reads were extracted and counted using featureCounts (v2.0.1) (Liao et al., 2014) and DESeq2 (v1.30.0) (Love et al., 2014) was used for differential expression analysis. Command lines for the processing steps are available in the supplementary file.