Dna gel extraction kit

The gut microbiota genomic DNA was extracted from oyster digestive tissues using the E.Z.N.A. soil DNA kit (Omega Bio-Tek, GA, USA). The DNA was quantified by NanoDrop 2000 (Thermo Scientific, Wilmington, DE, USA). The hypervariable region V3-V4 of the bacterial 16S rRNA genes were amplified with primer pairs 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) using 2× Phanta Flash master mix (Vazyme, Nanjing, China) in a PCR thermocycler (Bioer Technology, Hangzhou, China). The PCR parameters were initial denaturation at 95°C for 3 min, followed by 30 cycles of denaturing at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 45 s, and single extension at 72°C for 10 min. The PCR product was purified using a DNA gel extraction kit (Omega Bio-Tek, GA, USA) and then pooled in equimolar and paired-end sequenced on an Illumina MiSeq PE300 platform/NovaSeq PE250 platform (Illumina, San Diego, USA).
The raw 16S rRNA gene sequencing reads were demultiplexed and quality filtered by fastp version 0.20.0 and merged by FLASH version 1.2.7. OTUs with 97% similarity cutoff were clustered using UPARSE version 7.1. The taxonomy of each OTU representative sequence was analyzed by RDP Classifier version 2.2 against the 16S rRNA database using a confidence threshold of 0.7.

The gut microbiota genomic DNA was extracted from oyster digestive tissues using the E.Z.N.A. soil DNA kit (Omega Bio-Tek, GA, USA). The DNA was quantified by NanoDrop 2000 (Thermo Scientific, Wilmington, DE, USA). The hypervariable region V3-V4 of the bacterial 16S rRNA genes were amplified with primer pairs 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) using 2× Phanta Flash master mix (Vazyme, Nanjing, China) in a PCR thermocycler (Bioer Technology, Hangzhou, China). The PCR parameters were initial denaturation at 95°C for 3 min, followed by 30 cycles of denaturing at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 45 s, and single extension at 72°C for 10 min. The PCR product was purified using a DNA gel extraction kit (Omega Bio-Tek, GA, USA) and then pooled in equimolar and paired-end sequenced on an Illumina MiSeq PE300 platform/NovaSeq PE250 platform (Illumina, San Diego, USA).
The raw 16S rRNA gene sequencing reads were demultiplexed and quality filtered by fastp version 0.20.0 and merged by FLASH version 1.2.7. OTUs with 97% similarity cutoff were clustered using UPARSE version 7.1. The taxonomy of each OTU representative sequence was analyzed by RDP Classifier version 2.2 against the 16S rRNA database using a confidence threshold of 0.7.

Seedlings were carefully removed from the matrix and root-adhering matrix particles (< 0.02 mm in diameter) were collected with a brush, chilled on ice immediately and then stored at -20°C for subsequent processing. DNA in the matrix samples was extracted using a modified CTAB method according to DNA extraction kit (Tiangen biotech (Beijing), Co. Ltd). The 16S rRNA genes were amplified [23 ] using the universal bacterial primers GC-338F (5’- CGCCCGGGGCGCGCCCCGGGGCGGGGCGGGGGCGCGGGGGGCCT- ACGGGAGGCAGCAG-3’) and 518R (5’- ATTACCGCGGCTGCTGG-3’) with the addition of a barcoding sequence. 16S rRNA amplification PCR reactions were performed using a Biometra T-gradient thermal cycler in a reaction volume of 50 μL, containing 3.2 μL of 2.5 mM dNTPs, 2.0 μL of 20 mM GC-338 F primer and 2.0 μL of 20 mM 518 R primer, 5.0 μL of 10× buffer, 0.4 μL rTaq, 50 ng template DNA, and variable volume ddH₂O. Before amplification, an initial denaturation step of 94°C for 5 min was performed, followed by 30 cycles of 94°C for 1 min, 55°C for 45 s, and 72°C for 1 min, and. a final elongation step at 72°C for 10 min. After resolving the amplicons by agarose gel electrophoresis, amplicons within the appropriate size range were cut from the gel and PCR products were purified using a DNA Gel Extraction Kit (Omega Bio-Tek) following the manufacturer’s protocol, prior to sequencing.