16s rRNA of lichen
The quality and concentration of the extracted DNA were measured using a NanoDrop spectrophotometer (ND-1000, NanoDrop Technologies, Wilmington, DE, United States). The V3V4 region of the bacterial 16S rRNA gene was amplified by PCR (95C for 5 min, followed by 25 cycles of 95C for 30 s, 50C for 30 s, 72C for 40 s, and 72C for 7 min) using the primers 341F (5- CCTAYGGGRBGCASCAG-3) and 806R (5-GGACTACNNGGGTATCTAAT-3) and a high-fidelity polymerase. Indexed adapters were added to the ends of the primers. The PCR products were mixed with the same volume of 2 loading buffer and were subjected to 1.8% agarose gel electrophoresis for detection. Samples with a bright main band of approximately 460 bp were chosen and mixed in equal-density ratios. Then, the mixture of PCR products was purified using a GeneJET Gel Extraction Kit (Thermo Fisher Scientific, Waltham, MA, United States). Sequencing libraries were generated using NEB Next Ultra DNA Library Prep Kit for Illumina (NEB, USA) following manufacturers recommendations and indexed adapterswere added. The constructed libraries were validated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, United States) and quantified with a Qubit 2.0 Fluorometer (Thermo Fisher). Finally, paired-end sequencing was conducted using an Illumina NovaSeq6000 PE250 (Illumina, Inc., San Diego, CA, United States) at CD Genomics, Co., Ltd. (NY, USA).