16S rDNA sequencing of the lung: adult male
A specific primer with barcode was synthesized to amplify 16s rDNA V4-V5 region. The primer sequence was 515F-5, GTGCAGCMGCCGCGG-3, 907R-5 CCGTCAATTCMTTTRAGTTT-3'. Briefly, the PCR reaction consisted of 5 FastPfu Buffer 4ldNTPs (2.5 mM) 2 lForward Primer(5M) 0.8 lReverse Primer (5M) 0.8 lFastPfu Polymerase 0.4 lTemplate DNA 10 ng, and then Supplemented  ultra-pure water to 20 l. The reaction conditions were as follows: 5 min at 95C, 27 cycles (30s each at 95C and 53C respectively and 45s at 72) and 10min at 72. The amplification products were analyzed by Illumina high-throughput sequencing (Illumina PE250).