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Microbial DNA was extracted from faecal samples of rats using the E.Z.N.A .® Soil DNA Kit ( Omega Bio-Tek, Norcross, GA, U.S.) following the manufacturer’s protocols. Two primers (338F(5’-ACTCCTACGGAGGCAGCAG-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT)) were used to amplify the hypervariable regions(V3 to V4) of the bacterial 16S ribosomal RNA gene. Polymerase chain reaction was performed with the following programme: 3 min of denaturation at 95?; 27 cycles at 95? for 30 s, 55? for 30 s, and 72? for 45 s, and a final extension at 72? for 10 min. Purified amplicons were pooled in equimolar amounts and paired-end sequencing (2x300) was carried out on the Illumina MiSeq platform (Illumina, San Diego, USA). |
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