Microbial DNA and RNA Extraction and Sequencing

Biomass sampling was performed when switching incubation pressure (Fig. 1C). Total microbial genomic DNA was extracted and purified using the modified SDS-based method described by Natarajan et al.^{43 (link)}, and stored at −20 °C before further assessment. The quantity and quality of extracted DNA were measured using Qubit 4.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) and agarose gel electrophoresis, respectively. The extracted microbial DNA was processed to construct metagenome shotgun sequencing libraries with insert sizes of 350 bp following the standard Illumina TruSeq DNA Sample Preparation Guide. Each library was sequenced by Illumina NovaSeq 6000 platform (Illumina, USA) with PE150 strategy at Shanghai Personal Biotechnology (Shanghai, China). The extraction of RNA from sediment samples was carried out using the RNeasy® PowerSoil® Total RNA Kit (Cat. No. 12866-25, Qiagen, Germany) according to the manufacturer’s instructions, then quantified using a Qubit 4.0 Fluorometer (Invitrogen, Carlsbad, CA, USA). To ensure DNA removal, the RNA extracts were treated with TURBO DNase (Cat. No. AM2238, Invitrogen, Waltham, MA, USA) as directed by the manufacturer. The purified RNA was converted to cDNA, then the metatranscriptomic library was constructed by using Illumina TruSeq Stranded mRNA LT Sample Prep Kit, and subsequent sequencing as described above.