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The V3-V4 hypervariable regions of the bacteria 16S rRNA gene were amplified with primers 338F and 806R by thermo cycler PCR system (GeneAmp 9700, ABI, USA). The PCR reactions program was performed as follows: 3 min of denaturation at 95 °C, 27 cycles of 30 s at 95 °C, 30s for annealing at 55 °C, and 45s for elongation at 72 °C, and a final extension at 72 °C for 10 min. The PCR reactions were performed in triplicate 20 μL mixture containing 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 and 10 ng of template DNA. The PCR products were extracted from a 2% agarose gel and further purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA). Quantitation was done with QuantiFluor ™ -ST (Promega, USA) according to the manufacturer’s protocol. |
RNA-Seq |
GENOMIC |
unspecified |
SINGLE
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