title: Amplicon seq of permafrost
design: DNA was extracted from the pre-thaw and post-thaw soil permafrost subsamples using the Qiagen PowerSoil Pro Kit (Qiagen, Hilden, Germany) following the manufacturers protocol. DNA was then amplified in 12 L reactions using PCR with the following primers and conditions: 515F with Nextera adaptors (5TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGYCAGCMGCCGCGGTAA) and  926R with Nextera adaptors (5GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCGYCAATTYMTTTRAGTTT) of the V4 regions of the 16S rRNA gene to identify bacterial and archaeal communities (Parada et al., 2016; Quince et al., 2011). The PCR reactions were set up as follows: 6 l DreamTaq Hot Start Green (Thermo Fisher Scientific, Waltham, MA, United States), 2.6 L of a sterile water solution containing 0.5 g/l Bovine serum albumin (BSA) to reduce potential PCR inhibitors (Kreader, 1996), 0.7 L forward primer (5 M), 0.7 L reverse primer (5 M), and 2 L template DNA, for a 12 L reaction total. The thermocycler conditions for 16S rRNA amplification were as follows: enzyme activation at 94C for 3 min, 35 cycles of denaturation at 94C for 45 s, annealing at 50C for 60 s, and extension at 72C for 90 s, and lastly a final extension at 72C for 10 min. PCR products were then visualized via gel electrophoresis. During each round of DNA extraction, we included two empty tubes to serve as blanks. All samples amplified successfully and were sent to the University of New Hampshire Hubbard Genome Center, Durham, NH for multiplexing and sequencing on an Illumina NovaSeq 6000 (Illumina, San Diego, CA, USA) with 2x250bp chemistry. A portion of the samples were resequenced to achieve acceptable sequencing depth (> 20,000 reads per sample). To control for run-to-run biases, several samples that were sequenced to sufficient depth on the first run were also resequenced on the second run.