RNA Seq of water bacteria in lake
The filter membranes containing bacteria and fungi were cut into pieces, and total DNA was extracted using the Mo Bio Soil DNA Extraction Kit (MoBio, Carlsbad, CA, USA). The DNA concentration and quality were measured using a Thermo NanoDrop 2000 UV spectrophotometer and verified by 1% agarose gel electrophoresis. After quality inspection, the DNA was amplified using bacterial 16S rDNA universal primers 515F (5-GTG CCA GCM GCC GCG G-3) and 907R (5- CCG TAA TTC MTT TRA GTT T -3) (Guo et al., 2020).Bacterial amplification was performed using a 4 L of 5FastPfu Buffer, 2 L of 2.5 mM dNTPs, 0.8 L of each primer (5 M), 0.4 L of FastPfu Polymerase (TransGen AP221-02: TransStart Fastpfu DNA Polymerase), 0.2 L of BSA, and 10 ng of DNA template. The PCR program consisted of an initial denaturation at 95 C for 3 minutes, followed by 27 cycles of 95 C for 30 seconds, 55 C for 30 seconds, and 72 C for 45 seconds, with a final extension at 72 C for 10 minutes. Products were detected by 2% agarose gel electrophoresis, purified using the Trelief Gel DNA Recovery Kit (Tsingke Biotechnology Co., Ltd., Beijing, China), and quantified using a DS-11 spectrophotometer (DeNovix, USA). After library construction with the NEXTflex Kit (Bioo Scientific, USA), paired-end sequencing was performed on the Illumina Nextseq2000 platform (Illumina, San Diego, USA).