Fecal Microbiome Sample Preparation

Fresh fecal material (between 10 and 50 grams per person) was collected in sterile containers and immediately manipulated and homogenized within a maximum of 3 hours from defecation. During the time between defecation to homogenization, samples were kept at 4°C. Thirty ml of RNAlater solution (Applied Biosystems, Foster City, CA) were added to 10 grams of sample and the mixture was homogenized in sterile bags, using a stomacher apparatus (IUL Instruments, Barcelona, Spain) (three cycles at high speed, one minute per cycle). Homogenized samples were then stored at -80°C until use. For DNA extraction, samples were thawed and the QIAamp DNA Stool Mini kit was used (Qiagen Ltd., Strasse, Germany), as previously described [16 (link)].