Sample Preparation for Transcriptome Analysis and RNA-Seq Analysis

Three replicates of wild type (H66) and deletion strain (Δs479) were cultured in Hv-Ca medium supplemented with uracil at 45°C and grown to OD_{650 nm} = 0.6–0.7. Total RNA was isolated using NucleoZOL^TM (Machery and Nagel), and RNA samples were sent to Vertis Biotechnologie AG (Martinsried, Germany) for further treatment. Total RNA was treated with T4 Polynucleotide Kinase (NEB) and rRNA depleted using an in-house protocol, and cDNA library preparation was preceded by ultrasonic fragmentation. After 3′ adapter ligation, first-strand cDNA synthesis was performed using 3′ adapter primer and 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/μl 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 fractionated (200–550 bp) by preparative agarose gel electrophoresis and sequenced on an Illumina NextSeq 500 system using 1 × 75 bp read length. TruSeq barcode sequences which are part of the 5′ TruSeq sequencing adapter are included in Supplementary Table 4. Sequencing reads are deposited at the European Nucleotide Archive (ENA) under the study accession number PRJEB41379. For data analysis, reads were mapped to the genome using bowtie2 (version 2.3.4.1) with the “–very-sensitive” option and defaults otherwise (Langmead et al., 2009). Then, reads per feature were counted using featureCounts (version 1.6.4) and analyzed for differential expression with DeSeq2 (version 1.2.11) (Liao et al., 2014; Love et al., 2014).