112 Chapter 4 unit of gene transcription. Aware of differing extraction and sequencing methods for T1 and T2, these two data points have only been used in separate analyses. TPM values were used for bubble plot generation with the packages ggplot on RStudio. All figures were layout edited using Adobe Illustrator CC v22.1. Reverse-transcription quantitative polymerase chain reaction (RTqPCR) Selected RNA samples were used for RT-qPCR (Figure 1) in order to confirm patterns emerging from metatranscriptomic analyses. Three primer pairs were selected to quantify transcription of genes of interest: hydrazine synthase subunit A, with hzsA-F (5”-WTCGGRTATCARTATGTAG-3”) and hzsA-R (5”- AAATGGYGAATCATARTGGC-3”), adapted from previously published primers (Harhangi et al., 2012); particulate methane monooxygenase subunit A, with pmoA-F (5”-SCGRGTRMAGCCSGGTGAGA-3”) and pmoA-R (5”- YGATGGYCCMGGYACMGAGT-3”), designed for this study; and methyl-coenzyme M reductase subunit A, with mcrA-F (5”-AAAGTGCGGAGCAGCAATCACC-3”) and mcrA-R (5”-TCGTCCCATTCCTGCTGCATTGC-3”) (Vaksmaa et al., 2017). Bacterial and archaeal 16S rRNA gene-targeting primers 16S-F (5”- AAACTYAAAKGAATTGRCGG”) and 16S-R (5”-ACGGGCGGTGWGTRC-3”) were used as housekeeping genes for data normalization, adapted from previously published primers (Engelbrektson et al., 2010). For each sample, 50 ng of RNA was used for the reverse transcription reaction using the RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) following manufacturer’s instructions. Each qPCR reaction consisted of 12 μL SYBR Green FastMix (QuantaBio, MA, USA), 1 μL forward and 1 μL reverse primer (10 μM working solutions), 10 μL DEPC-treated water (Thermo Fisher Scientific, Waltham, MA, USA) and 1 μL template single stranded cDNA in a total volume of 25 μL. The qPCR program consisted of the following steps: initial denaturation (94 ºC for 5 min), denaturation (94ºC for 30 sec), annealing (temperature as below for 30 s), elongation (72ºC for 30 s), and melting curve (50-95ºC, 0.5ºC increase per 5 s). Denaturation, annealing and elongation steps were repeated for 40 cycles. Annealing temperatures for hzsA, pmoA, mcrA and general 16S rRNA gene primers were 55ºC, 62ºC, 62ºC and 55ºC, respectively. To check for DNA contamination qPCR was performed directly on RNA samples. This confirmed that DNA contamination was neglectable, given that all CT values
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