Fastdna spin soil kit

Microbial DNA was extracted from raw silage samples using a FastDNA SPIN Soil Kit (MP Biomedicals, Santa Ana, CA, USA). In brief, silage (approximately 0.3 -0.4 g wet weight for each sample) was rst added to a 2 ml lysing matrix tube added with homogenizing reagent provided by kit, then employed a series of lysing, homogenizing, and DNA puri cation and elution procedure following the manufacturer's instructions. DNA quality was checked by 1% agarose gel electrophoresis and spectrophotometry (NanoDrop 2000, Thermo Scienti c, Waltham, MA, USA; 260 nm/280 nm optical density ratios). PCR was used to amplify the variable (V3-V4) region of the 16S rRNA genes using 12-bp barcoded primers (forward 338F: 5'-ACTCCTACGGGAGGCAGCA-3'; reverse 806R: 5'-GGACTACHVGGGTWTCTAAT-3'). PCR products were extracted from a 2% agarose gel and puri ed with a AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA). Finally, the cloned libraries were pooled in equimolar amounts and sequenced on the Illumina MiSeq PE300 platform (Illumina Corporation, San Diego, CA, USA) at Shanghai Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China).
The sequence data for all samples were deposited in the NCBI Sequence Read Archive (SRA) under BioProject number PRJNA522947.

Microbial DNA was extracted using a FastDNA SPIN Soil Kit (MP Biomedicals, Santa Ana, CA) following manufacturer's instructions. DNA quality was checked by 1% agarose gel electrophoresis and spectrophotometry (NanoDrop 2000, Thermo Scienti c, USA; 260 nm/280 nm optical density ratios). PCR was used to amplify the variable (V3-V4) region of the 16S rRNA genes using 12-bp barcoded primers (forward 338F: 5'-ACTCCTACGGGAGGCAGCA-3'; reverse 806R: 5'-GGACTACHVGGGTWTCTAAT-3'). PCR products were puri ed with the Trans PCR Puri cation Kit and cloned with the Ultra DNA Library Prep Kit for Illumina (New England Biolabs Inc., Ipswich, MA, USA). Finally, the cloned libraries were pooled in equimolar amounts and sequenced on the Illumina MiSeq PE300 platform (Illumina Corporation, San Diego, USA) at Shanghai Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) .
Sequence data of all samples were deposited in the NCBI Sequence Read Archive (SRA) under the BioProject number PRJNA522947.

DNA was extracted using the Fast DNA SPIN Soil kit (MP Biomedicals, Solon, OH, United States) and quantified using the NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States). The fungal internal transcribed spacer (ITS) region was double-end sequenced on the Illumina MiSeq platform (San Diego, CA, United States). PCR amplification of the 16S rRNA gene was conducted using the primers 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 926R (5′-CCGTCAATTCMTTTGAGTTT-3′). The sequences of the primers were ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2 (5′-GCTGCGTTCTTCATCGATGC-3′) (Amato et al., 2013) (link). Trans Start Fast Pfu DNA polymerase was used for PCR amplification in the GeneAmp 9700 PCR system (Applied Biosystems, Foster City, CA, United States). Each 20-μl reaction system contained 0.8 μl of DNA (final concentration, 10 ng), 4 μl of 5× FastPfu buffer, 2 μl of 2.5 mM dNTPs, 0.8 μl of each primer (5 μM), 0.4 μl of FastPfu polymerase, 0.2 μl of BSA, and sterile double-distilled water to a final volume of 20 μl. The thermal cycling conditions were as follows: pre-denaturation at 95 °C for 3 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 45 s. A final extension at 72 °C for 10 min was also included.