Hiseq2500 pe250

Microbial DNA was extracted from ruminal fluid samples using the E.Z.N.A. stool DNA kit (Omega Bio-tek, Norcross, GA, United States) according to the manufacturer’s protocols. The 16S rDNA V3–V4 region of the eukaryotic ribosomal RNA gene was amplified by PCR (95°C for 2 min, followed by 27 cycles at 98°C for 10 s, 62°C for 30 s, and 68°C for 30 s and a final extension at 68°C for 10 min) using primers 341F: CCTACGGGNGGCWGCAG and 806R: GGACTACHVGGGTATCTAAT, where the barcode is an eight-base sequence that is unique to each sample. PCRs were performed in a triplicate 50 μL mixture containing 5 μL of 10 × KOD buffer, 5 μL of 2.5 mM dNTPs, 1.5 μL of each primer (5 μM), 1 μL of KOD polymerase, and 100 ng of template DNA.
Amplicons were extracted from 2% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, United States) according to the manufacturer’s instructions and quantified using QuantiFluor-ST (Promega, United States). Purified amplicons were pooled in equimolar ratios and paired-end sequenced (2 × 250) on an Illumina platform by Hiseq2500 PE250 according to the standard protocols. The raw reads were deposited into the NCBI Sequence Read Archive (SRA¹) database (Accession Number: SRP151436).

0.25 g F. taipaiensis rhizospheric soil sample was used for DNA extraction by CTAB method²⁷ . The genomic DNA after diluted to 1 ng μl^-1 was amplified by PCR via high-fidelity DNA polymerase and Phusion® High-Fidelity PCR Master Mix with GC Buffer (New England Biolabs) by using specific primer 515F (5′-GTGCCACCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) for the bacterial 16sRNA gene V4 sequence, the amplification condition was pre-denaturation at 98 °C for 2 min, denaturation at 98 °C for 30 s, annealing at 50 °C for 30 s, extending at 72 °C for 1 min, after 30 cycles, and then extending 72 °C again for 5 min to end the PCR reaction. The PCR products were tested by 2% agarose gel electrophoresis and then were extracted by gel extraction kit (Qiagen). The DNA library was constructed by using TruSeq® DNA PCR-Free Sample Preparation Kit (Illumina) and then was sequenced by HiSeq2500 PE250.

Microbial genomic DNA was extracted from rat colonic feces using the QIAamp DNA stool mini kit for DNA according to the manufacturer’s instructions. The hypervariable V3-V4 region of the bacterial 16S rRNA gene was analyzed. All DNA samples were amplified and purified using primers pair (338F and 806R). Finally, the high-throughput sequencing was performed on the Illumina HiSeq platform by HiSeq2500 PE250 at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China).

On day 0 and day 10, a 500-ml seawater sample was collected from each bucket with or without the olivine addition. The collected seawater was firstly filtrated through a 20-μm nylon sieve and then sequentially filtrated through 3- and 0.2-μm pore size polycarbonate membrane filters (47-mm diameter; Millipore) for microbe collection at a negative pressure of <0.01 MPa. The bacteria that settled on the membrane filter of 3-μm pore size were defined as the particle-attached fraction (PA, 3–20 μm), while those that settled on the filter of 0.2-μm pore size were defined as the free-living fraction (FL, 0.2–3 μm). The DNA of bacteria from both the 3- and 0.2-μm filters were extracted using a DNeasy PowerSoil Kit (Qiagen, Germany) according to the manufacturer’s protocol.
Amplicon sequencing of the microbial community in the DNA extraction was performed by Tianjin Novogene Bioinformatic Technology Co., Ltd. (Tianjin, China). The PCR products of one replicate of the olivine-added group in surface seawater are not enough for sequencing, so this part of the data is excluded from the data processing. The V3–V4 region of the bacterial 16S rDNA gene was amplified using the primer pair 341F (5′-CCTAYGGGRBGCASCAG-3′) and 806R (5′-GGACTACNNGGGTATCTAAT-3′) (Zhang et al., 2016 (link)), and sequencing was performed on an Illumina Hiseq 2500 pe250.

Bacterial genomic DNA was extracted from ileal digesta using NucleoSpin^® DNA Stool kit (MACHEREY-NAGEL Company, Germany). The quality of DNA samples were checked with gel electrophoresis. Bacterial 16S rDNA sequences spanning the variable regions V3–V4 were amplified according to the method described previously (Wang et al., 2019 (link)). The sequencing of the PCR products was performed on an Illumina HiSeq2500 PE250 platform (Illumina, San Diego, CA, United States). The sequencing results has been submitted to the Sequence Read Archive of the NCBI (Accession no. SAMN13610217). All the effective reads were clustered into operational taxonomic units (OTUs) based on a 97% sequence similarity. Classification of OTUs at various taxonomic levels were implemented by comparing sequences to the GreenGene database. Microbial α-diversity was analyzed using the MOTHUR v1.31.2 program (Schloss et al., 2009 (link)). The principal co-ordinates analysis (PCoA) and plot and non-metric multidimensional scaling (NMDS) were used to assess pairwise distances among samples (β-diversity). Linear discriminant analysis (LDA) combined effect size measurements (LEfSe) was applied to identify the relative richness (LDA > 2, P < 0.05) of bacteria between groups.

DNA was extracted from biopsy specimens using the QIAamp DNA minikit (Qiagen, CA, USA). Details of amplification, sequencing, raw data processing, and differential taxon selection were described previously (7 (link)). Briefly, the V3-V4 region of microbial 16S rRNA gene was amplified using universal primers (341F, 5′-CCTACGGGNBGCASCAG-3′; 805R, 5′-GACTACNVGGGTATCTAATCC-3′) and sequenced on the Illumina HiSeq 2500 PE250 platform. Sequence reads were processed and clustered into operational taxonomic units (OTUs) using IMNGS (www.imngs.org). Differential microbial taxa with relative abundances of >1% after eradication were selected by paired t tests when P values adjusted by FDR were less than 0.05.