Ampure xt beads

Illumina RNA-Seq libraries were prepared with the TruSeq RNA Sample Prep kit (Illumina, San Diego, CA) per the manufacturer’s protocol. For the bacterial RNA-enriched sample, the polyA isolation step was omitted. The total RNA sample was prepared from all RNA present in the sample, while the polyA-selected library had the polyA isolation step performed. Adapters containing seven nucleotide indexes were ligated to the double-stranded cDNA. The DNA was purified between enzymatic reactions and the size selection of the library was performed with AMPure XT beads (Beckman Coulter Genomics, Danvers, MA). The libraries were pooled and sequenced on an Illumina MiSeq or Illumina HiSeq sequencer paired end run, as specified in the text.

The omics analysis was supported by LC-Bio Technology Co., Ltd, Hangzhou, Zhejiang Province, China. E.Z.N.A. ^®Stool DNA Kit (D4015, Omega, Inc., USA) was applied to extract DNA. Then, the V3-V4 region of the 16S rRNA gene was amplified with primers 341F (5’-CCTACGGGNGGCWGCAG-3’) and 805R (5’-GACTACHVGGGTATCTAATCC-3’) (Logue et al., 2016 (link)). AMPure XT beads (Beckman Coulter Genomics, Danvers, MA, USA) and Qubit (Invitrogen, USA) were used for the purification and quantification of the polymerase chain reaction (PCR) products respectively. Finally, the Illumina NovaSeq platform was used for DNA sequencing.

Fresh fecal material was collected in a sterile container, and 30 mg was placed in another sterile container. All samples were immediately stored at −80°C until further processing. DNeasy PowerSoil Pro Kit was used to isolate microbial genomic DNA from fecal samples (QIAGEN, Germany), and the isolation procedures were performed according to the manufacturer’s instructions. The total DNA was eluted in 50 μl of elution buffer and stored at −80°C until used for PCR. PCR products were confirmed by 2% agarose gel electrophoresis. Throughout the DNA extraction process, ultrapure water, instead of a sample solution, was used as a negative control to exclude the possibility of false-positive PCR results. Polymerase chain reaction (PCR) amplification of the bacterial 16S rRNA genes V3-V4 region was performed using the universal primers 338F and 806R with 30 cycles. The PCR products were purified with AMPure XT beads (Beckman Coulter Genomics, Danvers, MA, USA) and quantified by Qubit (Invitrogen, Waltham, MA, USA). Amplicon pools were prepared for sequencing, and the size and quantity of the amplicon library were assessed using an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, MA, USA) and Library Quantification Kit for Illumina (Kapa Biosciences, Woburn, MA, USA), respectively. The libraries were sequenced using the NovaSeq PE250 platform.

The hypervariable V3–V4 region of the bacterial small subunit (16S) rRNA gene was amplified by polymerase chain reaction (PCR) using the forward primer 341F:5’-CCTACGGGNGGCWGCAG-3’ and 805R:5’-GACTACHVGGGTATCTAATCC-3’). The amplified products were recovered and purified. The PCR products were purified using AMPure XT beads (Beckman Coulter Genomics, Danvers, MA, USA) and quantified using Qubit (Invitrogen, USA). The amplicon pools were sequenced and the sizes of the amplicon libraries were evaluated using an Agilent 2100 Bioanalyzer (Agilent, USA) and an Illumina Library Quantitation kit (Kapa Biosciences, MA, USA), respectively. All samples were sequenced on an Illumina Novaseq6000 PE250 platform (provided by Hangzhou Lianchuan Biological Information Technology Co., Ltd), as recommended by the manufacturer.

Metatranscriptomic sequencing libraries were generated from up to 1 μg of RNA using the Zymo‐Seq RiboFree™ Total RNA Library Kit (Zymo Research, USA), following the manufacturer's protocol, except that AMPure XT beads (Beckman Coulter, USA) were used in place of Select‐a‐Size MagBeads. Libraries were dual indexed with the Zymo‐Seq™ UDI Primer Plate Kit (Zymo Research, USA) following the manufacturer's protocol, purified twice using 0.8–1.2 volumes of AMPure XT beads and quantified using the Agilent 2200 TapeStation with D1000 HS screen tapes (Agilent Technologies, USA). Libraries were sequenced on the Illumina® NovaSeq™ 6000 platform (Illumina Inc., USA) using 300 cycle paired‐end chemistry.

We extracted DNA from different samples using Omega Bio-Tek (Soil DNA Kit D5625, United States), amplified the V4 region of the bacterial 16S rRNA gene using 515F (5’-GTGYCAGCMGCGGTAA-3′) and 806R (5’-GACTACHVGGGTWTCTAAT-3′) primers, and then amplified the ITS2 region of fungi using ITS1FI2 (5’-GTGARTCATC GAATCTTTG-3′) and ITS2 (5’-TCCTCTTATTGC-3′) primers. PCR amplification was performed in a 25 μl reaction mixture containing 50 ng template DNA, 12.5 μL Phusion Hot start flex 2X Master Mix, and 2.5 μL forward and reverse primers, and ddH₂O was used to adjust the volume to 25 μL. The PCR amplification conditions were as follows: initial denaturation at 98°C for 30 s, followed by 32 cycles consisting of denaturation at 98°C for 10 s, annealing at 54°C for 30 s, and extension at 72°C for 45 s. The bacteria and fungi were amplified 35 times and 32 times, respectively, followed by a final extension step at 72°C for 10 min. PCR products were purified and quantified using AMPure XT beads (Beckman Coulter Genomics, Danvers, MA, United States) and Qubit (Invitrogen, United States), respectively. Amplicon library was prepared, and its size and quantity were assessed using an Agilent 2,100 Bioanalyzer (Agilent, United States) and the Library Quantification Kit for Illumina (Kappa Biosciences, Woburn, MA, United States). Finally, we used the NovaSeq PE250 platform to sequence the library.

The DNA concentration and purity were examined using NanoDrop2000 (Thermo Scientific, Waltham, MA, USA) and the DNA quality tested with 1% agarose gel electrophoresis. Primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACHVGGGTATCTAATCC-3′) were used for PCR amplification of the V3–V4 variable regions of 16S rDNA. PCR products were recovered with 2% agarose gel, purified with AMPure XT beads (Beckman, Brea, CA, USA), eluted with Tris–HCl, and detected with 2% agarose electrophoresis. The Quantifluor TM-ST system was used for quantitative detection. Qualified samples were used to construct a PE 2*300 library and sequenced following the standard operating procedures of the Illumina MiSeq Platform.