RNAseq of metagenomics
One hundred nanograms of total DNA were used to evaluate 16S ribosomal RNA (rRNA) sequence using the Illumina HiSeq platform (Illumina, San Diego, CA, USA). Briefly, 16S rRNA/18S rRNA/ITS genes of distinct regions (16SV4/16SV3/16SV3-V4/16SV4-V5, 18S V4/18S V9, ITS1/ITS2, Arc V4) were amplified using specific primers (e.g. 16S V4: 515F-806R, 18S V4: 528F-706R, 18S V9: 1380F-1510R, et. al) with the barcode.All PCR reactions were carried out with Phusion High-Fidelity PCR Master Mix (New England Biolabs, Ipswich, MA, USA). Samples between 400-450bp were chosen for further experiments. PCR products was mixed in equidensity ratios. The mixed PCR products were purified with Qiagen Gel Extraction Kit (QIAGEN Inc, Valencia, CA, USA). Sequencing libraries were generated using NEBNext UltraTM DNA Library Pre-Kit for Illumina (Illumina, San Diego, CA, USA), following the manufacturer 's recommendations and index codes were added.