Qiamp fast dna stool mini kit

Metagenomic DNA was extracted from fecal samples using the QIAmp Fast DNA Stool Mini kit (Qiagen, UK) with a modified protocol combined with a repeated bead beating method [33 (link)]. Briefly, 1 ml of lysis buffer was added to the stool sample in the bead-beating tube. The samples were homogenised with the lysis buffer using a mini beadbeater, incubated at 70 °C for 15 min with mixing at regular intervals, followed by another 15-min incubation at 37 °C, and then centrifuged at 4 °C for 5 min at 16,000 × g. The supernatant was then treated with ammonium acetate to remove impurities and incubated on ice for 5 min. This was followed by another centrifugation step at 16,000 × g at 4 °C for 10 min, and then, the DNA was precipitated by combining with an equal volume of isopropanol and stored overnight at − 20 °C. The next day, the DNA was pelleted by centrifugation at 16,000 × g at 4 °C for 15 min, washed with ethanol and Tris–EDTA, and treated with Proteinase K, RNAse, and purified according to the manufacturer’s instructions (QIAmp Fast DNA Stool Mini kit; Qiagen, UK). The DNA was quantified using Qubit and stored at − 30 °C until further use.

Total bacterial metagenomic DNA was extracted using a modified protocol which combined the repeat bead beating method [13 (link)] with the QIAmp Fast DNA Stool Mini kit (Qiagen, UK). DNA from the fresh sample was extracted within 4 hours of collection, while DNA from the snap and -80°C frozen samples was extracted after 7 days at -80°C. Briefly, 1ml of lysis buffer (500mM NaCl, 50mM Tris-HCl pH8.0, 50mM EDTA and 4% sodium dodecyl sulphate) was added to the bead beating tubes containing the faecal sample. Samples were homogenised for 3 mins at max speed using the Mini Beadbeater (BioSpec). Samples were incubated at 70°C for 15mins to lyse the cells. Following centrifugation the supernatant was removed and the bead beating steps repeated. Following pooling of the supernatant, samples were treated with 10M ammonium acetate (Sigma Aldrich, Ireland) and then the DNA was pelleted and washed with 70% ethanol. The DNA was then RNAse and proteinase K treated. Finally the DNA was washed using buffers AW1 and AW2 (QIAmp Fast DNA Stool Mini kit; Qiagen, UK) and eluted in 200 μl of ATE buffer. DNA was quantified using the Nanodrop 1000 (Thermo Scientific, Ireland).