Total RNA Sequencing for Transcriptome Analysis

For total RNA sequencing (RNA-seq), RNA was isolated as described in Text S1. Illumina system-compatible sequencing libraries were prepared essentially as described previously (50 (link)). RNA quantity and integrity were assessed using an Agilent 2100 Bioanalyzer (Agilent RNA 6000 Nano kit). Briefly, RNA samples were initially purified using magnetic beads (AMPure XP magnetic beads; Becton, Dickinson), rRNA was removed (Ribo-Zero rRNA removal kit; Illumina), and sequencing libraries were generated (KAPA stranded RNA-Seq kit; KAPA Biosystems) according to the manufacturer’s protocol. Illumina system-compatible adapters containing sample-specific 8-nucleotide-long barcoding sequences were ligated, and the cDNA libraries were PCR amplified. The resulting sequencing libraries were evaluated using an Agilent 2100 Bioanalyzer with a DNA 1000 chip and quantified by real-time PCR using the NEBNext Library Quant kit for Illumina (New England Biolabs). Raw sequencing data were generated on a NextSeq500 platform (Illumina) using paired-end 75-cycle sequencing run-compatible reagents (150 cycles, NextSeq 500/550 Mid Output v2 sequencing kit; Illumina). Libraries were prepared and sequenced in biological triplicates for each tested growth condition.