Qiaamp powerfecal dna kit

For B. anthracis strains, overnight cultures were diluted 100-fold into 25 ml LB and cultured for 8 h. Genomic DNA was extracted using QIAamp PowerFecal DNA Kit (QIAGEN, Hilden, Germany). For E. coli strains, overnight cultures were diluted 100-fold into 20 ml LB and cultured for 3 h. Induction was done by addition of aTc to 100 ng/ml for an hour before genomic DNA was extracted with QIAamp PowerFecal DNA Kit (QIAGEN, Hilden, Germany).
For DNA digestion experiments, 200 ng of genomic DNA was challenged by Fnu4HI, HaeIII, or MspI (New England Biolabs, Ipswich, MA, USA) at 37°C for an hour, followed by visualization using electrophoresis. All extractions and digestions were conducted in biological triplicate.

We performed all DNA isolations and library preparations in a UV-sterilized laminar flow hood to prevent aerosol contamination. We extracted total DNA from each guano sample using the QIAamp PowerFecal DNA Kit (MO BIO Laboratories, QIAGEN Co., Carlsbad, CA) following the manufacturer’s instructions with the following alterations: prior to homogenization, we incubated fecal samples in the provided lysis solutions (Powerbead + Solution C1) for 10 min at 70 °C. Next, we homogenized the fecal material in a Fisherbrand Bead Mill 24 homogenizer (Fisher Scientific, Pittsburgh, PA) at 6 m/s for 1–2 min, until the fecal slurry was fully homogenized. At the elution step, we eluted with warmed PCR-grade water instead of the provided C6 buffer and incubated columns for two minutes prior to centrifugation. In addition to our samples, we extracted one “blank” (water only) sample to account for bacterial contamination of the extraction kit, which has been documented as an important source of error in other metagenomic studies [57 (link), 58 (link)]. As a positive control, we also extracted 25 µL of genomic DNA from a mock microbial community of known composition (ZYMOBIOMICS, Zymo Research, Inc., Irvine, CA). Purified DNA extracts were preserved at − 25 °C prior to next generation sequencing (NGS) library preparation.

For each sample, we performed extractions using several fecal pellets (up to 0.25 g total). We extracted total DNA using the QIAamp PowerFecal DNA Kit (MO BIO Laboratories, Qiagen Co.) following the manufacturer's instructions with the following alterations; prior to homogenization, we incubated fecal samples in the provided lysis solutions for 10 min at 70°C. Next, we disrupted the fecal material in a Fisherbrand Bead Mill 24 Homogenizer (Fisher Scientific) at 6 m/s for 1–2 min, until the fecal slurry was fully homogenized. At the elution step, we eluted with 55°C PCR‐grade water and incubated columns for two minutes prior to centrifugation. In addition to our samples, we extracted one “blank” water sample per extraction kit. Purified DNA extracts were preserved at −25°C prior to library preparation.

DNA extraction was performed using QIAamp Power Fecal DNA Kit (QIAGEN, Germany). For samples immersed in solution, aliquots were centrifuged at 13,000 g for 5 min, and the supernatant was discarded. The pellet was washed with PBS, and centrifuged again at 13,000 g for 10 min. Subsequent DNA extraction steps were performed according to the manufacturer’s instructions. In addition, DNA concentration and the A260/280 ratio were tested by Colibri Microvolume Spectrometer (TIRERTEK BERTHOLD, Germany).

All stool samples were collected in Sun Yat-sen University Cancer Center, kept in sealed containers with some air at a temperature below 8 °C for transit, then processed within 24 hours after collection. Sample protector for DNA (Takara, 9750) was added in all processed samples and stored in a -80°C freezer. Total DNA was extracted from thawed fecal samples using the QIAamp PowerFecal DNA Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The concentration of genomic DNA in each sample was quantified using a NanoDrop 2000 spectrophotometer (Thermo Scientific). All qualified samples were stored at -20°C for further analysis.