Sparq puremag beads

Total genomic DNA was extracted from 250 mg soil using the DNeasy PowerSoil Pro DNA Kit (Qiagen, USA) following the manufacturer’s protocol. The DNA quantity was measured by a Nanodrop spectrophotometer (Thermo Fisher Scientific, USA). The 341F and 805R primers were used to amplify the V3-V4 region of the 16S rRNA gene for bacterial communities. PCR was processed in a 50 μl total volume by using 2× sparQ HiFi PCR master Mix (QuantaBio, USA) under the following conditions: initial denaturation for 2 min at 98°C; 28 cycles of denaturation for 20 sec at 98°C; annealing for 30 sec at 60°C; extension for 1 min at 72°C; and a final extension step for 1 min at 72°C. Subsequently, the amplicon was purified using sparQ Puremag Beads (QuantaBio, USA) and indexed using 5 μl of each Nextera XT index primer in a 50 μl PCR reaction using 10 cycles of the above described PCR conditions. PCR products were combined, purified using sparQ Puremag Beads (QuantaBio, USA), and pooled to produce a final loading concentration of 4 pM. Then, pooled libraries were sequenced using Illumina MiSeq (Illumina, USA) to generate paired-end reads in 2×250 bp using the 341F and 805R primers [14 (link)].

For NIH 3T3 ATAC-seq^{75 (link)}, 100,000 cells were used per experiment and for liver samples 3 mg of pulverized tissue was used as starting material. Following centrifugation at 500 g for 5 min at 4 °C, cells were washed with cold PBS and then resuspended in cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40). Cells were lysed on ice for 15 min and then spun down at 500 g for 10 min at 4 °C. After removing the supernatant, the nuclei were resuspended in transposition reaction mix (25 µl TD 2x reaction buffer, 2.5 µl Nextera Tn5 Transposase, 22.5 µl nuclease free water). The reaction was incubated for 30 min at 37 °C. DNA was purified using a PCR purification kit (ThermoFisher) and eluted in elution buffer (10 mM Tris buffer, pH 8). DNA was amplified for 10 cycles using a thermocycler and barcoded primers^{74 (link)}. DNA purification was achieved using sparQ PureMag Beads (Quantabio). Libraries were sequenced at the University of British Columbia (now SBME Sequencing Facility).

Metagenomic DNA was extracted from 130 mg of faeces using the QIAamp BiOstic bacteremia DNA kit (Qiagen) as previously described [4 (link)]. DNA was quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific) and metagenomic libraries were prepared using 50 ng of DNA and the NEBNext Ultra II DNA Library Prep Kit (New England BioLabs) as per the manufacturer’s recommendations. Size selection and purification of the metagenomic libraries was carried out using SparQ PureMag beads (Quantabio) and an Illumina Genome Analyzer (Illumina) with a KABA SYBR Fast Universal qPCR Kit (Kapa Biosystems) was used for quantification. The metagenomic libraries were normalized and pooled and 225 pM was sequenced together with PhiX (1 %) on a NovaSeq 6000 with a S4 flow cell (300 cycles) as per the manufacturer’s instructions. DNA from a mock community of 20 bacterial strains (MSA-2002; ATTC) was also extracted and sequenced as a positive control.