Five patients with INF isolates and CloNo isolates (nose isolates PFGE-identical to infection isolates) were selected (HD04, HD21, HD26, HD29, HD33)(Total number of RNA-Seq experiments n = 120). Randomly two INF isolates and two CloNo isolates were selected and grown in TSB overnight at 37°C under vigorous shaking in ambient air. Cultures were then diluted in TSB or 50% heat-inactivated, pooled human serum from 18 healthy individuals plus 50% PBS 1:500 and grown for 6h under the same conditions as before. Bacterial cells were then harvested, washed in PBS and immersed in RNAprotect (Qiagen, Hilden, Germany). Cells were pelleted after brief incubation. Cells were mechanically lysed with zirconia beads 3x20s on a tissue homogenizer (Precellys 12, Bertin, Montigny-le-Bretonneux, France) and RNA was extracted with the RNeasy mini kit (Qiagen, Hilden, Germany). RNA was quantified on a Qubit fluorometer (Thermo Fisher Scientific, Waltham, MD, USA). DNA was digested with DNA-free kit (Invitrogen, Karlsbad, CA, USA). Quality was assessed on a Bioanalyzer with RNA Nano kit (Agilent, Santa Clara, CA, USA) and only samples with a RIN >7.0 were accepted. rRNA depletion and strand specific cDNA sequencing on a HiSeq instrument (Illumina) were conducted at BGI genomics (Shenzhen, China). A mean of 11,061,832 paired-end, 150 nt Illumina reads were generated. Bases less than Q30 and adapter sequences of the reads were trimmed, and any reads shorter than 35 nt were removed using Trimmomatic version 0.36 [102]. Trimmed high-quality reads were then aligned to the assembly of isolate HD04-1 (CP052985, CP052986, and CP052987) with HISAT2 (version 2.1.0) [126]. Normalization and differential expression analyses were performed with DESeq2 [127]. The COG annotation was carried out with GAMOLA2 [109]. Volcano plots were graphed in R with the EnhancedVolcano package version 1.6.0 [128].

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