145 Physiological stress response to sulfide exposure of freshwater anaerobic methanotrophic archaea Germany), with initial manual pottering of samples to disrupt the granules. DNA and RNA quality were determined using a NanoDrop Spectrophotometer ND1000 (Isogen Life Science, Utrecht, Netherlands) and a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA), respectively. Concentrations were measured with a Qubit 2.0 fluorometer using the DNA dsDNA HS kit for DNA and RNA HS kit for RNA (Thermo Fisher Scientific, Waltham, MA, United States). For the T2 and T3 planktonic (filtrate fraction) DNA samples, the AMPpure XP (Beckman Coulter, CA, USA) DNA purification kit was employed. The planktonic fraction of time points T0 and T1 yielded insufficient DNA for downstream library preparations. All RNA samples included an RNAase-Free DNAase treatment (Qiagen, Hilden, Germany). Sequencing was performed by Macrogen Europe BV (Amsterdam, The Netherlands). Metagenomics Metagenomic sequencing was performed with a TruSeq DNA PCR free library using an insert size of 550bp on a NovaSeq6000 (Illumina) platform, producing 2x151bp paired-end reads (10 Gbp/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). Trimmed reads were co-assembled de novo using metaSPAdes v3.14.1 (Nurk et al., 2017) and mapped to assembled contigs using BBMap (BBTools v38.75) (Bushnell, 2014). Contigs at least 1,000-bp long were used as template for read mapping of metatranscriptomic sequences as well as for binning. Sequence mapping files were handled and converted using Samtools v1.10., later used for binning with CONCOCT v2.1 (Alneberg et al., 2014), MaxBin2 v2.2.7 (Wu et al., 2016), and MetaBAT2 v2.12.1 (Kang et al., 2019). Resulting metagenome-assembled genomes (MAGs) were dereplicated with DAS Tool v1.1.1 (Sieber et al., 2018) and taxonomically classified with the Genome Taxonomy Database Toolkit GTDB-Tk v2.1.0 (Chaumeil et al., 2019). Metagenomic mapping statistics were generated via CheckM v1.1.2 (Parks et al., 2015). For a metagenomic binning taxonomical read-recruitment assessment SingleM v0.16.0 (https://github. com/wwood/singlem) was employed. MAG completeness and contamination was estimated with CheckM2 v1.0.1 (Chklovski et al., 2023). Metagenome-assembled genomes were annotated with DRAM v1.0 (Shaffer et al., 2020), and with default options, except min_contig_size at 1,000 bp, and METABOLIC v4 (Zhou et al., 5
RkJQdWJsaXNoZXIy MTk4NDMw