title: Day 2 - 25C - bare 3, archaea
design: Sediment (0 - 10 cm) samples were stored at -80 C before extraction. Samples were defrosted and mixed before 0.25 - 0.5 g of sediment for DNA extraction following the protocols of PowerSoil DNA Isolation Kit (MO BIO Laboratories Inc., Carlsbad, CA, USA) and SurePrep Soil DNA Isolation Kit (Fisher BioReagents, Thermo Fisher Scientic, Fair Lawn, NJ, USA). Samples were then quantified by Qubit hsDNA High Sensitivity Assay Kit (Invitrogen, Carlsbad, CA, USA). The V3 and V4 region of bacterial 16S rRNA gene was amplified using the primers described by Klindworth et al. (2013) 314F (5-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3) and 785R (5' GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC), and the V4 region of archaeal 16S rRNA gene was amplified using the primers U519F (5-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCAGYMGCCRCGGKAAHACC-3) and 806R (5-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACTACNNGGGTATCTAAT-3) (Porat et al., 2010) using specific Illumina adapter overhang nucleotide sequences. For each sample, duplicate PCRs were run with a final volume of 40 l using Qiagen multiplex PCR master mix (QIAGEN, Valencia, CA), 4 l cDNA as DNA template and a final primer concentration of 0.3M. The PCR conditions for bacterial 16S rRNA genes were as follows: One initial polymerase activation and denaturation cycle at 95 C for 15 min, 30 cycles each at 95 C for 30 s, 55 C for 30 s, 72 C for 30 s, followed by final elongation step at 72 C for 5 min. The PCR conditions for archaeal 16S rRNA genes were as follows: One initial polymerase activation and denaturation cycle at 95 C for 15 min, 27 cycles each at 94 C for 15 s, 53 C for 45 s, 68 C for 45 s, followed by a final elongation step at 68 C for 5 min. For each sample, the duplicate PCR was pooled, visualized via 1 % agorose gel electrophoresis and cleaned with Agencourt AMPure XP magnetic bead system (Beckman Coulter, Brea, CA, USA). Following the manufacturer's protocol, the cleaned PCR products underwent an indexing PCR to add Nextera XT indexes (Illumina, Inc., Alliance Global FZ, Dubai, UAE). The Index PCR conditions were as follows: One cycle at 95 C for 15 min, 8 cycles each at 95 C for 30 s, 55 C for 30 s, 72 C for 30 s, followed by a final step at 72 C for 5 min. Successful amplification was visualized via 2 % agorose gel electrophoresis and another cleaning step with Agencourt AMPure XP magnetic bead system (Beckman Coulter, Brea, CA, USA) followed. The cleaned products were then quantified by Qubit hsDNA High Sensitivity Assay Kit (Invitrogen, Carlsbad, CA, USA), and pooled in equimolar ratios. Finally, the pooled product was then checked on the BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA) for size and presence of dimer primers. The sample was sequenced at 6 pM with 25% phiX on the Illumina MiSeq, 2 x 300 bp paired-end by MiSeq reagent Kit v3 (Illumina, Inc., Alliance Global FZ, Dubai, UAE)  according to the manufacturer's specifications at the Bioscience Core Lab facility at King Abdullah University of Science and Technology, Saudi Arabia.
identifiers.namespace: SUB6524074
identifiers.id: 8