Ds 11 nanodrop spectrophotometer

Bacterial suspension was harvested under anaerobic conditions and collected in ice-cold falcon tubes. The samples were quickly cooled down by placing them carefully in liquid nitrogen. Pelleting of cells was done by centrifugation at 10,200 × g at 4°C for 3 min. The supernatant was discarded, the pellet dissolved in sterile suspension buffer (3 mM EDTA, 200 mM NaCl, pH 8.0) and after mechanical disruption proceeded with an acid-phenol extraction of RNA (Nicolas et al., 2012 (link)). Quality of the RNA was analyzed on a 1.5% agarose gel and concentration was determined with NanoDrop DeNovix DS-11 Spectrophotometer (DeNovix, Wilmington, United States). RNA was extracted in quadruplicates from independent cultures.
Northern blot analysis was carried out as previously described (Troitzsch et al., 2021 (link)). The digoxigenin-labeled RNA probes were synthesized by in vitro transcription with T7 RNA polymerase using gene-specific primers (fliC-NB forw 5′-ATGAGAGTTAATACAAATGTAAGTGC-3′ and fliC-NB rev 5′-CTAATACGACTCACTATAGGGAGACTATCCTAATAATTGT AAAACTCC-3′) on chromosomal DNA as template. 10 μg of total RNA were separated on a 1.5% agarose gel per sample. Chemiluminescent signals were detected with a Lumi-Imager (Intas Science Imaging Instruments GmbH). Intensity signals of Northern Blot bands were quantified with Fiji (Schindelin et al., 2012 (link)).

DNA was extracted from rectal swabs and feces using the repeated bead beating method (Yu and Morrison, 2004 (link)) and the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany), according to manufacturer’s instructions. This method combines lysis by bead beading, DNA precipitation, RNA and protein removal, and DNA purification by QIAamp DNA Stool Mini Kit to obtain the maximum yield from fecal material samples. About 0.1 g of fecal samples and 500 μl of rectal swab solution was used as starting material for DNA extraction. The cells were lysed in Lysing Matrix B tubes prefilled with 0.1 mm silica beads (MP Biomedicals, Santa Ana, California, USA) using FastPrep-24™ (MP Biomedicals, Santa Ana, California, USA) at 5.5 m/s for 3 min (with intermittent cooling on ice in between after every minute). Following the protocol of Yu and Morrison (Yu and Morrison, 2004 (link)), RNA and protein were removed from the samples by DNase-free RNase (10 mg/ml, Qiagen, Hilden, Germany) and Proteinase K (Qiagen, Hilden, Germany) treatment. DNA was subsequently purified using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) as previously described (Yu and Morrison, 2004 (link)). DNA integrity and quantity were determined using a Nanodrop DeNovix DS-11 Spectrophotometer (DeNovix Inc., Wilmington, DE USA) according to manufacturer’s instructions.

Samples were bathed in liquid nitrogen and pulverized into a fine powder using a mortar and pestle. Approximately 100 µg of powdered sample was lysed with 700 µl of QIAzol lysis buffer (Cat # 79306, QIAGEN) and homogenized by passing the solution through QIAshredder spin columns (Cat # 79654, QIAGEN). RNA isolation was performed using standard miRNeasy mini kit (Cat # 217004, QIAGEN) according to the manufacturer’s protocol. Quality and quantity of the RNA samples were assessed using a DeNovix DS-11 nanodrop spectrophotometer (DeNovix, DE, US) and Agilent Bioanalyzer with the RNA6000 Nano Lab Chip (Agilent Technologies, Santa Clara, CA).

Samples were bathed in liquid nitrogen and pulverized into a fine powder using a mortar and pestle. Approximately 100 µg of powdered sample was lysed with 700 µl of QIAzol lysis buffer (Cat # 79306, QIAGEN) and homogenized by passing the solution through QIAshredder spin columns (Cat # 79654, QIAGEN). RNA isolation was performed using standard miRNeasy mini kit (Cat # 217004, QIAGEN) according to the manufacturer’s protocol. Quality and quantity of the RNA samples were assessed using a DeNovix DS-11 nanodrop spectrophotometer (DeNovix, DE, US) and Agilent Bioanalyzer with the RNA6000 Nano Lab Chip (Agilent Technologies, Santa Clara, CA).