RNA-seq Analysis of Streptococcus pneumoniae

RNA was extracted and prepared as above from biological quadruplicates of S. pneumoniae D39. RNA was pooled and analyzed on a Bioanalyzer 2100 (Agilent) to confirm a RIN value > 8 according to the manufacturer’s instructions. RNA was then submitted to the Australian Genome Research Facility (AGRF) for sequencing. In brief, the Epicentre Bacterial Ribozero Kit (Illumina) was used to deplete ribosomal RNA content before the generation of barcoded libraries using Ultra Directional RNA kit (New England Biolabs). Prepared libraries were then sequenced using an Illumina HiSeq2500 with Version 3 SBS reagents and 2 × 100 bp single-end chemistry. Reads were aligned to the S. pneumoniae D39 genome (GenBank accession number NC_008533) using BOWTIE2 version 2.2.6^{84 (link)}. Counts for each gene were obtained using SAMtools version 1.2^{85 (link)} and BEDtools version 2.24.0^{86 (link)}, and differential gene expression was determined using R (DESeq Library) version 3.2.2^{87 (link)}. Transcriptomic data have been deposited in the NCBI Gene Expression Omnibus databank under submission identifier GSE141681.