Total RNA Extraction

Total RNA was extracted using the RNeasy PowerMicrobiome Kit following the manufacturer’s instructions (Qiagen, Hilden, Germany) with some modifications with respect to the bead beating procedure. Frozen feces (250 mg) were weighed into 2-mL screw-cap tubes, containing 0.6 g sterile glass beads (Ø 0.1 mm), 0.4 g ceramic beads (Ø 1.4 mm), 0.55 g ceramic beads (Ø 2.8 mm) and 650 μL guanidinium thiocyanate buffer. TheFastPrep-24 instrument (MP Biomedicals, Heidelberg, Germany) was used for homogenization which consisted of three bead beating steps- each one 1 min at 6.5 m/swith cooling on ice between the bead beating steps. Thereafter, samples were processed according to the manufacturer’s instructions (Qiagen). To remove genomic DNA, the extracted RNA was treated with DNase I (Turbo DNA kit, Life Technologies Limited, Vienna Austria) following the manufacturer’s instructions. The elution volume was 50 μL. The total RNA of each sample was quantified by the Qubit 2.0 Fluorometer (Life Technologies Corporation, CA, United States) using Qubit RNA Assay Kit according to the manufacturer’s instructions as well as qualified with Agilent Bioanalyzer 2100 (Agilent Technologies, Waldbronn, Germany). The Agilent RNA 6000 Nano Assay (Agilent Technologies, Waghäusel-Wiesental, Germany) was used to determine the RNA integrity numbers (RIN), which ranged between 6 and 10. The cDNA synthesis was performed using the High Capacity Reverse Transcription Kit (Life Technologies Foster City, United States) following the manufacturer’s instructions. One μg of total RNA and 0.5 μL of RNase Inhibitor (Qiagen) were added to each reaction. One aliquot of the cDNA was sent for 16S rRNA gene amplicon sequencing, whereas a second aliquot was used for absolute quantification of total bacteria and virulence factors.