Ribo zero rrna removal kit for gram positive bacteria

RNaseq was essentially performed as previously described (Crosby et al., 2016 (link)). Briefly, cultures of LAC and LACΔagr were grown in TSB with DMSO alone or 100 μM apicidin (diluted in neat DMSO) in triplicate in a 48-well plate. Cultures were grown to optical density of 4 at 600 nm, and cells were harvested and treated with RNAProtect Bacteria Reagent (QIAGEN). Cells were lysed using lysostaphin and RNA purified using the RNeasy mini kit (QIAGEN). The samples were treated with DNA-free (Ambion) and sample quality was affirmed via Bioanalyzer (Agilent). Ribosomal RNA was depleted using Ribo-Zero rRNA Removal Kit for gram positive bacteria (Illumina). cDNA libraries were generated at the University of Iowa Genomics Division using the TruSeq Stranded mRNA Library Prep kit (Illumina). Sample were barcoded, pooled and sequenced in 125×125 paired-end reads on an Illumina HiSeq 2000 sequencer. The resulting reads were aligned to an updated S. aureus USA300 genome file containing annotations for small RNAs (Carroll et al., 2016 (link)) using SeqMan NGen (DNASTAR) and reads were quantified using ArrayStar (DNASTAR). Genes were considered differentially expressed if they showed a ≥ 4-fold change in expression with 95% confidence as evaluated by Student’s t test with a false discovery rate (FDR) correction applied for multiple t tests.

50 strains representative of the four major genetic subclades (Fig. 1a,b; Supplementary Table 7) were assayed in triplicate at mid-exponential and early-stationary phases of growth. RNA was extracted with the RNeasy kit (Qiagen) following the manufacturer’s instructions. rRNA was depleted using the Ribo-Zero rRNA removal kit for Gram-positive bacteria (Illumina), as described previously^{93 ,108} . The quality of the total RNA and rRNA-depleted RNA was evaluated with RNA Nano, and Pico chips, respectively (Agilent Technologies), and an Agilent 2100 Bioanalyzer. The cDNA libraries were prepared with indexed reverse primers from the ScriptSeq Index PCR primers kit (Illumina), and purified with AMPureXP beads (Beckman Coulter). The quality of the cDNA libraries was evaluated with High-Sensitivity DNA chips (Agilent Technologies). For each sample, the cDNA library concentration was measured fluorometrically with Qubit™ dsDNA HS assay kits (Invitrogen). The cDNA libraries were diluted, pooled, and sequenced with an Illumina NextSeq instrument. This same protocol was used for the comparative RNA-seq analysis of the 9T and 10T isogenic strains (Supplementary Table 15).