title: 16S rRNA of marine microbial community
design: RNA extraction was conducted using zymoBIOMICS DNA/RNA miniprep kit following the manufacturers instructions. The extracted RNA was then purified and converted to cDNA using SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen) according to Wemheuer et al.. The quality and quantity of cDNA were measured through a NanoDrop device (Thermo Scienti_c Inc., Wilmington, DE, USA). Only the high-quality samples (A260/230 > 1.7 and A260/280 > 1.8) were used for further analysis. To determine microbial community structure, we amplified the V3-V4 region (forward primers 5-CCTACGGRRBGCASCAGKVRVGAAT-3 and reverse primers 5-GGACTACNVGGGTWTCTAATCC-3) of bacterial 16S rRNA gene of cDNA samples by PCR procedure. Quantified amplicons were sequenced by the Illumina MiSeq platform (Illumina, San Diego, CA, USA). We filtered the sequence contained poly-N strings, retaining the sequence length longer than 200 bp. Processed sequences were identified at a sequence similarity cut off of 0.97 using VSEARCH (1.9.16) and Silva database as reference database. The RDP classifier (Ribosomal Database Program) was used to determine community composition of each sample.