Nebnext ultra dna sample preparation kit

The main protocol of faecal bacterial DNA extraction has followed the procedure described by Hao et al. [34 (link)]. 16S rRNA gene amplicon preparation and sequencing were as same as the description in the previous study [36 (link)]. Briefly, DNAs were extracted using HiPure Stool DNA Kits (Magen, Guangzhou, China). The qualities of the DNA were appraised using a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The extracted DNA was amplified by PCR with the KAPA HiFi Hotstart ReadyMix PCR kit (KAPA Biosystems, Wilmington, MA, USA). The V3–V4 region of the bacterial 16S rRNA gene was amplified using primers F341 (5′-ACTCCTACGGGRSGCAGCAG-3′) and R806 (5′-GGACTACVVGGGTATCTAATC-3′) [37 (link)]. The amplicons were gathered from 2% agarose gels and purified with a QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). Purified DNAs were re-quantified using an Agilent DNA 1000 Kit (Agilent Technologies, Waldbronn, Germany). Library quality was assessed on a Qubit 2.0 Fluorometer (Life Technologies, Grand Island, NY, USA). Sequencing library preparation was done using NEBNext ultra DNA sample preparation kit (New England Biolabs Inc., Ipswich, MA, USA). Then, reads of approximately 250–300 bp paired-end were sequenced on the Illumina MiSeq platform.

For DNA extraction, 1 g of mixed rumen contents was divided from the raw samples. DNAs were extracted using Qiagen’s DNA Extraction Kit™ (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The qualities of the DNA were appraised using a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The extracted DNA was amplified by PCR with the KAPA HiFi Hotstart ReadyMix PCR kit (Carlsbad Life Technologies, USA). The V3–V4 region of the bacterial 16S rRNA gene was amplified using primers F341 (5’-ACTCCTACGGGRSGCAGCAG-3’) and R806 (5’-GGACTACVVGGGTATCTAATC-3’) [21 (link)]. The PCR products were gathered from 2% agarose gels and purified with a QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). Purified DNAs were re-quantified using an Agilent DNA 1000 Kit (Agilent Technologies, Waldbronn, Germany). Library quality was assessed on a Qubit 2.0 Fluorometer (Life technologies, Grand Island, NY, USA). Sequencing library preparation was done using NEBNext ultra DNA sample preparation kit (NEB, USA) following the manufacturer’s protocol. Then, reads of approximately 250–300 bp paired-end were sequenced on the Illumina MiSeq platform.

Sequencing libraries were generated using the NEB Next Ultra DNA sample preparation kit (New England Biolabs, Ipswich, MA, USA) following the standard Illumina sample-preparation protocol, and index codes were added. The quality of the library was assessed using a Qubit 2.0 Fluorometer (Life Technologies, Grand Island, NY) and an Agilent Bioanalyzer 2100 system (Agilent Technologies, Palo Alto, CA). The library was sequenced on an Illumina HiSeq platform, and single 150 bp × 2 paired-end reads were generated.