) together with 23 unrelated barcoded samples. This resulted in 10,276,620 paired-end reads (2 × 100 bp) for sample 307.14, encapsulated and 8,715,247 paired-end reads (2 × 100 bp) for sample 307.14, nonencapsulated. De novo assembly The reads of the variants 307.14 nonencapsulated and 307.14 encapsulated were subjected to buy Trichostatin A de novo assembly using SPAdes (version 2.4.0, kmer sizes = 33,55,67,81,91,93,95,97,99)

[58]. Only scaffolds equal or longer than 500 bp were used for the further analyses. The assembly of 307.14 nonencapsulated resulted in 2088272 bp in 63 scaffolds and a n50 of 79979 bp. The assembly of 307.14 encapsulated resulted in 2083495 bp in 69 scaffolds and a n50 of 71589 bp. Polymorphisms detection To detect assembly errors, for the assemblies of the strains 307.14 nonencapsulated and 307.14 encapsulated a remapping was performed using bowtie2 (version 2.0.0beta6, options: -N 1 –very-sensitive) [59]. Differences

were detected using samtools (version 0.1.19, mpileup). To detect polymorphisms between the two strains, the reads of 307.14 nonencapsulated were mapped to the de novo assembly 307.14 encapsulated and vice versa. The mapping was performed using bowtie2 (version 2.0.0beta6, options: -N 1 –very-sensitive). Subsequently, polymorphisms of both mappings were determined using samtools (version 0.1.19, mpileup) [60]. Gene expression assays Microarray Bacteria were cultured

as described for the adherence and invasion assay to mid-logarithmic phase in CDM, Selonsertib mouse 5.5 mM glucose, pH 7. Double volume of RNAprotect® bacteria reagent (Qiagen, Germany) was added to the bacterial suspension to stop further transcription. The samples were vortexed, incubated for 5 min at room temperature and then centrifuged at 4500 × g for 10 min at +4°C. The RNA was extracted with the RNeasy® Mini Kit (Qiagen) following the manufacturer’s instructions using a Mickle vibratory tissue disintegrator (Mickle Laboratory Engineering Company Ltd., UK) for mechanical disruption of the bacteria. Contaminating DNA was removed using the DNA-free™ Kit (Life Technologies) as described by the manufacturer. RNA purity, concentration and quality/integrity were checked using with the Interleukin-2 receptor NanoDrop® spectrophotometer ND-1000 (Thermo Scientific, USA) and the RNA Nano 6000 kit for the Agilent 2100 bioanalyzer (Agilent Technologies, USA) following the manufacturer’s instructions. The entire transcriptome was analyzed by microarray as follows. RNA samples were hybridised to the BμG@S SPv1.4.0 microarray designed by the Bacterial Microarray Group at St. George’s, University of London and manufactured on the Agilent PI3K inhibitor SurePrint platform (Agilent Technologies). Labelled cDNA was prepared from 1 μg total RNA using Cy3-dCTP (GE Healthcare, UK) and SuperScript II reverse transcriptase with random hexamer primers (Life Technologies).