110 Chapter 4 we also performed PCR on RNA extractions for rt-qPCR and did not observe any PCR products. Thus, validating the effectiveness of the DNAase treatment in trace DNA removal for all RNA extractions employed. DNA and RNA concentrations were measured with a Qubit 2.0 fluorometer using the dsDNA and RNA HS Kits (Thermo Fisher Scientific, Waltham, MA, USA). DNA and RNA quality were determined using a NanoDrop Spectrophotometer ND-1000 (Isogen Life Science, Utrecht, The Netherlands) and a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA), respectively. Metagenomic libraries were prepared using the Nextera XT Kit (Illumina, San Diego, California, USA) following manufacturer’s instructions. Enzymatic tagmentation was performed with 1 ng DNA, followed by the incorporation of indexed adapters and amplification of the library. Amplified DNA libraries were then purified, and their quality and concentration were determined as aforementioned. Libraries were sequenced on an Illumina MiSeq platform (San Diego, California, USA) using the MiSeq Reagent Kit v3 (San Diego, California, USA), generating 300 bp pairedend reads. RNA samples of T1 and T2 were rRNA-depleted with MICROBexpress Kit (Thermofisher, Waltham, USA) and MEGAclear kit (Ambion, Life Technologie, Carlsbad, CA USA). Subsequently, 0.1 - 4 µg of RNA from T1-T3 were used to construct strand specific RNA-Seq libraries. Non-rRNA in RNA-Seq libraries were enriched by selective priming during the first strand cDNA synthesis reaction, as well as in the final library construction steps using TruSeq Stranded mRNA sample preparation guide (Illumina proprietary catalog RS-122-9004DOC). RNA from these samples was sequenced with an Illumina MiSeq platform (Illumina, CA, USA), generating 151 bp single-end reads. Metatranscriptomic libraries of T0, T4 and T5 were constructed using TruSeq stranded mRNA library Kit (Illumina, San Diego, California, USA). These RNA-Seq libraries were sequenced with an Illumina NovaSeq 6000 platform, generating 151 bp paired-end reads (San Diego, California, USA). All metatranscriptomes were triplicate RNA extractions and sequencing. Metagenomic and metatranscriptomic analyses Metagenomic data were analyzed as follows. Read quality was assessed with FASTQC v0.11.8 before and after quality-trimming, adapter removal and contaminant filtering, performed with BBDuk (BBTools v38.75). Trimmed reads were co-assembled de novo using MEGAHIT v1.2.911 (Li et al., 2016) and mapped
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