RNA-Seq of R. conorri and host
Total RNA isolation was carried out using Tri-Reagent (Molecular Research Center) following an optimized version of the manufacturers protocol. At each time point, culture medium was aspirated off carefully and the cells (HMECs or AAE2) infected with R. conorii were lysed in Tri-Reagent and processed for RNA isolation using our standard laboratory protocol. Total RNA thus obtained was subjected to DNase I treatment to eliminate genomic DNA contamination, purified by precipitation with 3M sodium acetate pH 5.5 (Ambion) and glycogen (5mg/mL) (Ambion), and dissolved in nuclease-free water. The quality of total RNA preparations was assessed on a Bioanalyzer (Agilent Technologies) and only samples with an RNA integration number (RIN) score of 9.0 were used for sequencing. RNA samples were enriched for bacterial coding and non-coding transcripts using Dynabeads Oligo (dT)25 (ThermoFisher Scientific) and Ribo-Zero Gold rRNA Removal Kit (Epidemiology) (Illumina) to remove mRNAs (eukaryotic) and rRNAs (bacterial, eukaryotic, and mitochondrial), respectively. Enriched RNA from each biological replicate was then split into two equal aliquots, of which one was treated with Terminator 5-Phosphate-dependent Exonuclease (TEX) to remove processed transcripts and designated as +TEX. The other untreated aliquot containing both primary (5-triphosphate) and processed (5-monophosphate) transcripts was designated as -TEX. Complementary DNA libraries for each sample were prepared independently using TruSeq RNA Sample Prep Kit following manufacturer instructions (Illumina). Strand-specific, paired end reads of 100 bases in length were sequenced on an Illumina HiSeq 1500.