Axygen pcr cleanup kit

DNA and RNA were extracted from the same filters using the All-Prep DNA/RNA Minikit (Qiagen). DNA was used to verify the overall community including dead and dormant cells at two time points 12 hours apart on either side (T₁ and T₄). RNA was converted to cDNA using the High Capacity Reverse Transcription Kit (Applied Biosystems). The V4 region of 18 S rRNA gene and rRNA (from cDNA) was amplified using the eukaryote specific primers E572 (forward) and E1009 (reverse), see Comeau et al.^{66 (link)}, coupled with a MiSeq© specific linking primer. To decrease potential PCR bias, 1, 5 and 10-fold diluted template was used for PCRs for each sample. The PCR products of the three dilutions were pooled together and purified using the Axygen® PCR cleanup kit (Axygen) and then quantified spectrophotometrically with the Nanodrop 1000™ (ThermoFisher Scientific). Unique pairs of barcodes (tags) were added to the sample amplicons using the TruSeq® and Nextera® (both Illumina) barcode sets in a nested PCR as described in Comeau et al.^{67 (link)}. Equimolar concentrations of the sample barcoded amplicons were sequenced on the Illumina MiSeq at the Plate–forme d’Analyses Génomiques (IBIS, Université Laval, Québec, QC, Canada). All reads are deposited in NCBI GenBank Sequence Read Archive (SRA) under the BioProject number PRJNA383398 (GenBank: SRX2745611 to SRX2745642).

For the sequencing, the method described by Hoggard et al., 2018 [38 (link)] was followed. A purification step was conducted for the initial PCR reaction using an Axygen PCR cleanup kit (Axygen), and then the quality was verified with 1% Thermo Fischer Scientific Massachusetts, U.S. agarose gel electrophoresis. The purified solution was diluted; then, it was used in a range of 50 to 100-fold as a new template for a second PCR under similar conditions to the first PCR, except for using 10 cycles as recommended by Al-Sadi and Kazerooni, 2018 [39 (link)]. For this PCR round, the Illumina Nextera PCR primers described in Table 3 were used, which were followed by quantification with a Quantus^® by Promega (Promega, Quito, Ecuador)). Amplicons were pooled and submitted for sequencing using an Illumina MiSeq (Illumina, Inc., San Diego, CA, USA).

One milliliter of biomass was sampled and centrifuged at 7440 × g for 2 min. The approximately 0.25 g pellet was transferred with an aseptic stainless steel spoon to extract the total genomic DNA using the Power Soil DNA isolation kit (MoBio Laboratories Inc., USA) according to the manufacturer’s instruction manual.
The 16S rRNA gene of the bacteria was amplified using a universal primer pair 8F/1492R49 (link)50 (link). The PCR amplification of the NC10 phylum pmoA gene was performed using primer pairs A189_b/cmo682 and cmo182/cmo568, as previously described51 (link). Briefly, the PCR mixtures (25 μL) contained 1 μL of template DNA, 1 μL of each primer, 9.5 μL of RNAase-free water (Takara, Japan), and 12.5 μL of Ex Taq premix (Takara, Japan) according to the manufacturer’s instruction manual. The PCR program consisted of an initial denaturation at 94 °C for 3 min, 35 cycles of denaturation at 94 °C (1 min), annealing (55 °C and 2 min for 8F/1492R; 60 °C and 1 min for A189_b/cmo682; 62 °C and 1 min for cmo182/cmo568) and extension at 72 °C (2 min for 8F/1492R; 1 min for A189_b/cmo682 and cmo182/cmo568), and a final extension at 72 °C for 10 min. The obtained PCR products were purified using agarose gel electrophoresis and Axygen PCR Cleanup kit (Axygen Scientific Inc., CA, USA). The detailed information regarding the PCR primers used above is given in Table S1.