172 Chapter 6 with the RNeasy PowerSoil Kit (Qiagen, Hilden, Germany), with an initial manual pottering of samples to disrupt the granules. RNA extractions were stored at -70ºC. Metatranscriptomics-directed RNA samples were DNAase I-treated at 37°C for 30 min with the solutions included in the RiboPure Bacteria Kit (Thermo Fisher Scientific, Waltham, MA, United States). For RNA samples used for RT-qPCR, we employed the same DNAase-treatment as the one suggested in the reverse transcriptase (RT) cDNA synthesis kit - the Revert Aid H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, United States). DNA and RNA quality were determined using a NanoDrop Spectrophotometer ND-1000 (Isogen Life Science, Utrecht, Netherlands) and a Bioanalyzer 2100 (Agilent, Santa Clara, CA, United States), respectively. Concentrations were measured with a Qubit 2.0 fluorometer using the DNA dsDNA HS for DNA and RNA HS kit for RNA (Thermo Fisher Scientific, Waltham, MA, United States). Sequencing was performed by Macrogen Europe BV (Amsterdam, The Netherlands). Bacterial and archaeal 16S rRNA gene amplicon analysis Gene amplicon sequencing was performed on a MiSeq (Illumina) platform, using library kit Herculase II Fusion DNA Polymerase NEXTERA XT Index kit V2 (Illumina, Eindhoven, Netherlands), generating 2x300bp paired-end reads. See Supplementary Table 1 for bacterial and archaeal primers employed. Recovered 16S rRNA gene amplicon raw sequences were processed in R Studio version 2022.12.0+353 and R v4.2.2 with: DADA2 package v1.26.0, ggplot2 v3.4.0, phyloseq v1.42.0, vegan v2.6.4, DESEQ2 1.38.2, dendextend v1.16.2, tidyr v1.2.1, viridis v0.6.2, reshape v0.8.9, zoo v1.8.11 and plyr v1.8.8. First, we filtered out all flanking adapters attached to our primers with cutadapt v1.18 (Martin, 2011). Briefly, we used the DADA2 pipeline (Callahan et al., 2016) quality plots to trim forward and reverse bacterial reads at 270bp and 200bp whereas archaeal reads at 280bp and 200bp, respectively. Trimming was combined with trimLeft option 20 for primer removal. After error models were generated, sequences were dereplicated, merged and chimeras were discarded producing between 48,000-62,000 and 90,000-120,000 paired-end non-chimeric merged bacterial or archaeal sequences, respectively. Later, amplicon sequencing variants (ASV) were inferred and assigned using the Silva database release v.138.1(Quast et al., 2013). ASVs were clustered by taxonomy
RkJQdWJsaXNoZXIy MTk4NDMw