16S amplicon sequencing of adult human lung tissue
The bacterial 16S rDNA V4 region was amplified using the universal primer that target this region in most bacteria (Forward primer: 5-GTGCCAGCMGCCGCGGTAA-3 and Reverse primer: 5-GGACTACHVGGGTWTCTAAT-3) with the barcode. All Polymerase Chain Reactions were carried out with 15 L of Phusion High-Fidelity PCR Master Mix (New England Biolabs), 0.2 M of forward and reverse primers, and 10 ng template DNA. Thermal cycling consisted of initial denaturation (98 for 1 minutes), 30 cycles of denaturation (98 for 10 seconds), annealing (50 for 30 seconds), elongation (72 for 30 seconds), and finally 72 for 5 minutes. Same volume of 1X loading buffer was mixed with PCR products and electrophoresed on 2% agarose gel for detection. PCR amplicons were mixed in equidensity ratios. Then, Qiagen Gel Extraction Kit (Qiagen, Germany) was used to purify the PCR amplicon mixtures. TruSeq DNA PCR-Free Sample Preparation Kit (Illumina, USA) with index codes was used to generate sequencing libraries following manufacturer's instructions.