title: RNA-Seq of water fungi in lake
design: 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 fungal ITS genes with ITS1F (5- CTT GGT CAT TTA GAG GAA GTA A  3) and ITS2R (5- GCT GCG TTC TTC ATC GAT GC  3) (Guo et al., 2020).The fungal amplification system consisted of a 20 L reaction mixture containing 10 L of 10 Buffer, 2 L of 2.5 mM dNTPs, 0.8 L of each primer (5 M), 0.2 L of rTaq Polymerase (TaKaRa, Japan), 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).