DNA sequence from air sample (PTFE membrane, RNA isolation)
The RNA was isolated from PTFE membrane using RNeasy PowerWater Kit (Qiagen, Germany). The RNA was transcribed into cDNA using the EliZyme OneS Kit (Elizabeth Pharmacon, Czech Republic). The forward primer 5.8S_Fun 5-AACTTTYRRCAAYGGATCWCT-3 and reverse primer ITS4_Fun 5-AGCCTCCGCTTATTGATATGCTTAART-3 were used. The first PCR mix contained nuclease-free water, 400 nM of each of the appropriate primers, 25 L of EliZyme OneS kit Mix, 2.5 L of RTase, and 5 L of template in a total reaction volume of 50 L where each sample was run in 3 replicates. The thermocycling program was as follows: 10 min of reverse transcription at 50C, 2 min of denaturation at 95C, followed by 40 cycles of 10 s at 95C, 10 s at 50C for 16S rRNA, and 15 s at 72C; the final extension was run at 72C for 5 min. Next, the replicates were merged and concentrated using the commercial kit Genomic DNA Clean & Concentrator (ZYMO Research, Irvine, CA, USA). The second PCR reaction mix contained nuclease-free water, 1 M of each of the appropriate primers, 0.02 U/l KAPA HiFi HotStart ReadyMix and 1 l of template in a total reaction volume of 25 l. The following thermocycling program was used: 5 min of denaturation at 95 C, followed by 13 cycles of 20 s at 98 C, 15 s at 50 C and 15 s at 72 C. Final extension was run at 72 C for 5 min. Next, amplicons were sent to the Core Facility for Nucleic Acid at the University of Alaska Fairbanks for Illumina MiSeq platform sequencing.