16S rRNA human gut metagenome
Microbial DNA was extracted from 0.25 g of fecal sample using the repeated bead-beating protocol (Yu and Morrison, 2004) with only a few modifications (DAmico et al., 2021). In short, stool samples were resuspended in 1 mL of lysis buffer in the presence of four 3-mm glass beads and 0.5 g of 0.1-mm zirconia beads (BioSpec Products, Bartlesville, OK, USA), and bead-beaten in a FastPrep homogenizer (MP Biomedicals, Irvine, CA, USA) at 5.5 movements/s for 1 min three times. After 15-min incubation at 95C, supernatants were separated by centrifugation at 13,000 rpm for 5 min and incubated with 260 L of 10 M ammonium acetate and then with one volume of isopropanol for 30 min. Nucleic acid pellets were washed with 70% ethanol and resuspended in 100 L of TE buffer. RNA was removed by 15-min incubation at 37C with 2 L of DNase-free RNase (10 mg/mL). For the subsequent DNA purification steps, the DNeasy Blood and Tissue Kit (QIAGEN, Hilden, Germany) was used. DNA concentration and quality were assessed using the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA).The V3-V4 hypervariable regions of the 16S rRNA gene were amplified using the 341F and 785R primers with Illumina adapter overhang sequences, as previously described (DAmico et al., 2021). PCR products were purified using a magnetic bead-based system (Agencourt AMPure XP; Beckman Coulter), followed by sample indexing through a limited-cycle PCR with Nextera technology. Indexed libraries were purified with another clean-up step as described above and pooled at the equimolar concentration of 4 nM. Sequencing was performed by loading denatured and 5 pM diluted libraries into an Illumina MiSeq platform, using the 2  250 bp paired-end protocol per manufacturers instructions (Illumina, San Diego, CA, USA).