Qiaamp mini spin column

Briefly, a frozen aliquot (~100 mg) of each sample was suspended while frozen in a solution containing 500 μL extraction buffer (200 mM Tris, pH 8/200 mM NaCl/20 mM EDTA), 200 μL 20% SDS, 500 μL phenol:chloroform:isoamyl alcohol (25:24:1), and 500 μL 0.1 mm-diameter zirconia/silica beads (BioSpec Products). Microbial cells were lysed by mechanical disruption with a bead beater (BioSpec Products) for 2 minutes, after which two rounds of phenol:chloroform:isoamyl alcohol extraction were performed. DNA was precipitated with ethanol and resuspended in 50 μL TE buffer with 100 μg/mL RNase. Isolated DNA was subjected to additional purification with QiaAmp mini spin columns (Qiagen).

DNA was isolated from murine fecal pellets or patient fecal samples using 0.1-mm zirconia/silica beads (BioSpec Products) to homogenize the sample and phenol/chloroform/isoamyl extraction as previously described (Becattini et al., 2017 (link)). After phenol/chloroform/isoamyl extraction, DNA was precipitated in ethanol, resuspended in TE buffer with 200 µg/ml RNase, and further purified with QIAamp mini spin columns (Qiagen). The V4–V5 region of the 16S rRNA gene was amplified with the primers 563F (5′-nnnnnnnn-NNNNNNNNNNNN-AYTGGGYDTAAAGNG-3′) and 926R (5′-nnnnnnnn-NNNNNNNNNNNN-CCGTCAATTYHTTTRAGT-3′), where uppercase Ns represent 12-bp Golay barcodes and lowercase Ns represent additional nucleotides to offset the sequencing of the primers (Caporaso et al., 2012 (link)). Amplicons were purified with the Qiaquick PCR Purification kit (Qiagen). The purified PCR products were quantified using Agilent Technologies 2200 tape station and pooled at equimolar amounts. The TruSeq DNA library preparation kit (Illumina) was used according to the manufacturer’s instructions to prepare the library. The library was sequenced on an Illumina MiSeq platform following the manufactures instructions and a paired-end 250 × 250-bp kit.