Pneumococcal Gene Expression Analysis

For gene expression analysis, pneumococcal strains were grown to an OD₅₉₀ of 0.15 to 0.18 in triplicate. Five milliliters of cells was added to 1 ml of an ice-cold 95% ethanol–5% phenol solution, before centrifugation at 4,000 rpm for 10 min. The supernatant was removed, and the pellets were stored at −80°C until processing. RNA was extracted using the Maxwell 16 LEV simplyRNA cells kit (Promega, USA) and the Maxwell 16 LEV instrument (Promega, USA). The manufacturer’s protocol was followed from step 4, and the manufacturer’s lysis steps (steps 1 to 3) were replaced with the following protocol to improve cell lysis: pellets were resuspended in 50 μl TE with 3 mg/ml lysozyme and incubated at 37°C for 10 to 20 min. RNAseq analysis was done by using a MiSeq desktop processor (Illumina, USA) and the ScriptSeq complete kit (bacteria) (CamBio, UK), which includes an rRNA depletion step. Raw data were trimmed using Trimmomatic-0.32 and aligned using BWA-Mem and samtools. Expression data were generated using Rockhopper v2.0.3 (44 (link)) with D39 as the reference genome (GenBank accession number NC_008533.2).