RNA extraction, library preparation, and sequencing of the Foc transcriptome

Total RNA from three independent cultures grown under each condition was extracted separately using the TRIzol Reagent (Invitrogen) following the manufacturer’s instructions, then treated with RNase-free DNase I (TaKaRa, China). One part of total RNA from three independent cultures for each condition were pooled in equal amounts for RNA-seq analysis, and the other part was stored at −80° for further real-time quantitative PCR (qRT-PCR) verification. The quality and quantity of the total RNA was validated using agarose gel electrophoresis and a 2100 Bioanalyzer (Agilent Technologies). A total of 3 μg high-quality RNA (RNA integrity number > 8) per sample was prepared to generate cDNA libraries. Sequencing libraries were generated using the NEBNext Ultra RNA library prep kit for Illumina (New England BioLabs). The cDNA libraries were then sequenced on the Illumina HiSequation 2000 platform using a 100 bp paired-end sequencing strategy at the Novogene Corporation, Beijing, China.