177 Osmoregulation in freshwater anaerobic methane-oxidizing archaea under salt stress platform, generating 100-bp paired-end reads with 10 Gb throughput/sample. Raw sequences were quality trimmed using sickle v1.33 (https://github.com/najoshi/ sickle) and ribosomal RNA contaminant-filtered, mapped against the DRAMgenerated scaffolds and Transcripts per Million (TPM)-values generated using trancriptm v0.2 (https://github.com/sternp/transcriptm). To visualize an annotated or categorized gene expression profile, we employed the R package ggpubr. For “Ca. Methanoperedens” specific individual gene expression contribution, new TPM values were calculated only including “Ca. Methanoperedens” contigs. Metaproteomics Metaproteomic analysis was performed as recently described (Kleikamp et al., 2023; Pabst et al., 2022). For protein extraction and proteolytic digestion, ~ 100 mg of each cell pellet (wet weight) was dissolved in 175 µL 50 mM TEAB buffer (with 1% NaDOC) and 175 µL B-PER buffer (Thermo Scientific, Germany) by vortexing. Then acid washed glass beads (105–212 µm, Sigma Aldrich) were added and the mixtures were vortexed thoroughly. Thereafter, the samples were spun down and the supernatant was collected and proteins were precipitated by adding 1 volume TCA to 4 volumes supernatant. The obtained protein pellets were once washed with ice cold acetone and then dissolved in 6 M urea (in 100 mM ammonium bicarbonate, ABC). Further, the disulfide bridges were reduced by the addition of 10 mM DTT and reduced using 20 mM IAA. 200 mM ABC buffer was then added to the samples to obtain a solution with <1 M urea. Finally, proteolytic digestion was performed by adding trypsin (0.1 μg/μL in 1 mM HCl, Sequencing Grade Modified Trypsin, Promega) at a ratio of 50:1 (w:w, protein:trypsin) to the sample. The proteolytic digestion was performed overnight at 37°C, under gentle shaking at 300 rpm. Peptides were desalted using an OASIS HLB solid phase extraction well plate (Waters, UK) according to the instructions of the manufacturer, speed vac dried and stored at -20°C until further processed. Shotgun proteomic analysis followed with ~ 500 ng of proteolytic digest and analysed using an EASY nanoLC 1200, equipped with an Acclaim PepMap RSLC RP C18 separation column (50 μm x 150 mm, 2 μm), and a QE plus Orbitrap mass spectrometer (Thermo Fisher Scientific, Germany). The flow rate was maintained at 350 nL/min over a linear gradient from 5% to 25% solvent B over 88 minutes, and finally to 55% B over 60 minutes. Data were acquired from 0 to 175 min. Solvent A was H2O containing 6
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