Miseq sequencing system

Total RNA extraction was performed using TRIzol reagent (Invitrogen, MA). Six pairs of degenerative NP samples (100 mg each) and matched controls were tested. An Agilent 2100 Bioanalyzer (Agilent, CA) was utilized to examine RNA integrity (RIN). The library construction was performed using an Illumina TruSeq small RNA Sample Prep kit (Illumina, CA) and 1 μg of total RNA following the manufacturer’s instructions. Fifteen cycles of PCR were carried out. The quality of the RNA product was examined with a High Sensitivity DNA Chip and an Agilent 2100 Bioanalyzer (Agilent Technologies, CA), and the concentration was assessed by qPCR using a KAPA Library Quantification kit (KAPA Biosystems, CA). Each library was adjusted to a concentration of 20 pM and was sequenced with a MiSeq Reagent kit v3 for 150 cycles by an Illumina MiSeq Sequencing System (Illumina). An efficiency of 25 million sequence reads per flow cell was achieved.

The 16S rRNA gene ranged from V3 to V4 variable region was used as the target for bacterial community investigation by Illumina Miseq sequencing. PCR amplication primer was used according to Klindworth et al. [34 (link)] study and the protocal of library preparation guideline of Illumina. In general, PCR was performed by using KAPA HiFi HotStart ReadyMix kit (Kapa, Biosystems). Each PCR reaction contained genomic DNA 2.5 μl, forward and reverse primers 5 μl respectively, and KAPA mixture 12.5 μl. The amplication procedure was based on our previous study, and then the products were purified with AMPure XP magnetic beads (Beckman, Coulter), quantified using Qubit fluorometer (Invitrogen, Life Technologies). The secondary PCR amplication was performed to add the Illumina Nextera barcodes, using i5 and i7 primers following the manufacturer’s instruction, and then the purification process was executed again to remove nontarget fragments. Finally, the amplicons were normalized, pooled and sequencing was conducted using Illumina Miseq sequencing system (Illumina, SanDiego, USA).

Hypervariable regions (V3–V4) of the 16 S rRNA gene were amplified by PCR in a total volume of 50 μl containing 0.25 μl of BioFact F-Star Taq DNA polymerase (BioFACT™, Seoul, Republic of Korea), 20 ng of DNA template, 5 μl of 10× Taq buffer (20 mM Mg²⁺), 1 μl of 10 mM dNTP mix, and 2 μl of forward and reverse barcoded primers (10 pmol/μl). PCR was performed in a GeneAmp® PCR system 9700 (Applied Biosystems, Foster City, CA, USA) under the following thermocycling conditions: initial denaturation at 94 °C for 5 min, followed by 28 cycles of denaturation (30 s, 95 °C), annealing (30 s, 60 °C), and extension (30 s, 72 °C), a final extension step at 72 °C for 10 min, and cooling to 4 °C. PCR products were confirmed by electrophoresis in 1% agarose gels, visualized using a Gel Doc system (BioRad, Hercules, CA, USA), purified with PureLink Quick Gel Extraction and PCR Purification Combo Kit (Invitrogen, Carlsbad, CA, USA), and quantified using a Qubit 2.0 fluorometer (Invitrogen). The size of the libraries was assessed using BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA). The amplicons of participants were sequenced in an Illumina MiSeq sequencing system (Illumina, San Diego, CA, USA).

Total DNA was extracted from feces and SI contents using QIAamp DNA stool mini kits (Qiagen) in accordance with the manufacturer’s instructions. For bacterial PCR amplification, primers targeting 341 F and 805 R were used. The amplified product was purified and sequenced by Chunlab (Seoul, South Korea) with an Illumina Miseq Sequencing system (Illumina). The processing of raw reads started with a quality check and filtering of low-quality (60 (link) After chimeric filtering, reads that were not identified to the species level (with <97% similarity) in the EzBioCloud database were compiled. Operational taxonomic units with single reads (singletons) were omitted from further analysis. The alpha diversity (Shannon index) and beta diversity for the sample difference were estimated. A taxonomic cladogram was generated using LEfSe with a threshold of 2 on the logarithmic LDA score.^{61 (link)} A relationship based on a Pearson correlation between gut microbiota and SCFAs was visualized using Calypso software.^{62 (link)}

PCR reactions targeting the V4 region of the 16s rRNA gene consisted of 10 μL of GoTaq^® Colorless Master Mix 2× (Promega, Madison, WI, USA), 0.3 μM forward primer (515F: GTGCCAGCMGCCGCGGTAA), 0.3 μM reverse primer (806R: GGACTACHVGGGTWTC), 20 ng of genomic DNA, and 20 μL of water and were run on a Veriti Thermal Cycler (Applied Biosystems, Foster City, CA). These reactions were run with an initial denaturation at 94 °C for 3 min, 29 cycles of 94 °C for 45 s, 50 °C for 1 min, 72 °C for 1 min and 30 s, followed by a final extension of 10 min at 72 °C. PCR products were then checked on a 2% agarose gel.
For library preparation, PCR triplicates of each sample were pooled in an aliquot and purified using Agencourt AMPure XP magnetic beads (Beckman Coulter, Brea, CA, USA). Libraries were quantified by real-time PCR using a KAPA Library Quantification Kit for Illumina sequencing (Kapa Biosystems, Wilmington, MA, USA). Samples were then normalized to 3 nM prior to sequencing on an Illumina MiSeq sequencing system (Illumina, Inc., San Diego, CA, USA) at BPI Biotecnologia (São Paulo, Brazil). Raw sequences are accessible under BioProject ID PRJNA580001 in the NCBI Sequence Read Archive.

Metagenomic DNA was extracted with three different techniques each from a 0.5 g soil sub-sample and pooled (De León-Lorenzana et al., 2017 (link)). As such, 1.5 g soil from each plot (n = 3), treatments (n = 12) and sampling day (n = 5) was extracted for metagenomic DNA. Overall, 270 g soil was extracted and 180 DNA samples were obtained.
The V3-V4 hypervariable regions of 16S rRNA genes were amplified with DNA polymerase (Thermo Fisher Scientific, Hudson, NH, USA) in a final 25 μl reaction volume. Amplification was done with the PCR Thermal Cycler Multigene Optimax (Labnet, Edison, NJ, USA). The following thermal cycling scheme was used: initial denaturation at 94 °C for 10 min; 25 cycles of denaturation at 94 °C for 45 s, annealing at 53 °C for 45 s, and extension at 72 °C for 1 min; followed by a final extension at 72 °C for 10 min, using the forward primer 341F (5′-CCTACGGGIGGCWGCAG-3′) and the reverse primer 805R (5′-GACTACHVGGGTATCTAATCC-3′) containing the Illumina platform adapters and eight bp barcodes. All samples were amplified in triplicate, pooled in equal volumes and sequenced by Macrogen Inc. (DNA Sequencing Service, Seoul, Korea) with paired-end Illumina MiSeq sequencing system (Illumina, San Diego, CA, USA).

Tobacco (25 g) was suspended in 500 ml of sterile saline and shaken for 2 hr at 200 rpm, after which the supernatant was centrifuged at 10,000 g for 20 min. Genomic DNA was extracted from the resulting pellet using an EZNA® Soil DNA Kit (Omega). The genomic DNA was sent to GENEWIZ Inc. for PCR amplification and sequencing of the V3‐V4 hypervariable region of 16S rRNA genes (primers: 5′‐CCT ACG GRR BGC ASC AGK VRV GAA/T3′ and 5′‐GGA CTA CNV GGG TWT CTA ATC C‐3′) and the V7‐V8 hypervariable region of 18S rRNA genes (forward primers containing the sequence: 5′‐CGW TAA CGA ACG AG‐3′ and reverse primers containing 5′‐AIC CAT TCA ATC GG‐3′).
DNA libraries, validated by Agilent 2100 Bioanalyzer (Agilent) and quantified by Qubit 2.0 Fluorometer (Invitrogen), were multiplexed and loaded on an Illumina MiSeq sequencing system according to manufacturer's instructions (Illumina). Sequencing was performed using a 2 × 250 paired‐end (PE) configuration. Image analysis and base calling were performed using the MiSeq Control Software (MCS) of the MiSeq instrument.