16S-Seq of marine sediment: Liaohe estuary
The V3-V4 regions of bacterial 16S rRNA from each sample was amplified using primers 341F-806R (341F: ACTCCTACGGGAGGCAGCAG, 806R: GGACTACHVGGGTWTCTAAT) (Berg et al., 2012). All PCRs were carried out in 15 L reactions with 7.5 L Phusion High-Fidelity PCR Master Mix (New England Biolabs), 1 L of dNTPs (2.5 mM), 1 L of forward and reverse primers (10 M), and 1 L template DNA. Thermal cycling consisted of initial denaturation at 95 C for 2 min, followed by 25 cycles of denaturation at 94 C for 5 s, annealing at 55 C for 30 s, and elongation at 72 C for 30 s. The PCR products of each sample were detected by electrophoresis on 1.5% agarose gel, and all samples were successfully amplified. Then, the obtained PCR products were purified, quantified, and mixed in equal amounts, respectively, to construct the sequencing libraries. Finally, the library quality was determined using Agilent Bioanalyzer 2100 system and Qubit 2.0 Fluorometer (Thermo Scientic), and then all libraries were sequenced on an Illumina NovaSeq platform.