173 Osmoregulation in freshwater anaerobic methane-oxidizing archaea under salt stress and relative abundance using the R package phyloseq (McMurdie & Holmes, 2013) and plotted with ggplot2 v3.4.0(Wickham, 2016). Quantitative PCR For the determination of gene abundances in DNA samples, all qPCR amplifications consisted of 12 µl SYBR Green FastMix (QuantaBio, Beverly, MA, United States), 0.6 µl of forward and reverse primer (both at 10 µM) topped up with DEPC-treated water (Thermo Fisher Scientific, Waltham, MA, United States) yielding a total volume of 25 µl. See Supplementary Table 1 for bacterial and archaeal primer set employed. The qPCR program consisted of the following steps: initial denaturation (94°C for 5 min), denaturation (94°C for 30 s), annealing temperature (varying-T-ºC for 30 s), elongation (72°C for 30 s), and melting curve (50–95°C, 0.5°C increase per 5 s). Denaturation, annealing, and elongation steps were repeated for 40 cycles. For the gene expression determination of genes encoding for osmolyte production enzymes, qPCR primer sets specific for the kamA and ablB genes of “Ca. Methanoperedens” were synthesized (Biolegio, Nijmegen, The Netherlands; primer sequences and optimized annealing temperature in Supplementary Table 1). In brief, synthesized cDNA was used for quantitative PCR with a C1000 Touch thermocycler coupled with a CFX96 Touch Real-Time PCR detection system (Bio-Rad Laboratories, the Netherlands) with the same PCR mixtures and program reported Dalcin Martins et al. (2022). For comparison of gene expression, we calculated ΔΔCt values between the genes of interest and the 16S rRNA gene copy abundance of archaea. PCR efficiencies were obtained based on amplification-slopes, keeping only amplifications with efficiencies above 90%. Single-copy bacterial or archaeal 16S rRNA gene-carrying pGEM-T Easy vector plasmids (with the same mentioned primer set) (Promega, The Netherlands) were used for quantification calibration. Metagenomics Metagenomic sequencing was performed with a TruSeq DNA PCR free library using an insert size of 350bp on a NovaSeq6000 (Illumina) platform, producing 2x151bp paired-end reads (10Gbp/sample). Read quality was assessed with FASTQC v0.11.9 before and after quality trimming, adapter removal, and contaminant filtering, performed with BBDuk (BBTools v38.75). We employed Kaiju v1.7.2 for read-based 6
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