AMPLICON sequencing of sediment metagenome
Co-extraction of DNA and RNA from 2 g of homogenized subsamples was carried out using the RNeasy Powersoil Total RNA Kit and RNeasy Powersoil DNA Elution Kit (QIAGEN, Germany). The extracted RNA was treated with OneStep PCR Inhibitor Removal Kit (Zymo Research, USA), and TURBO DNA-free Kit (Invitrogen, USA) and complete removal of DNA was confirmed by electrophoresis on 1% agarose gels. Five l RNA was reverse transcribed using the Transcriptor First Strand cDNA Synthesis Kit (Roche, Germany). PMA-treated DNA was extracted using the Powersoil Kit (QIAGEN, Germany). The V4 hypervariable region of the 18S rRNA gene was PCR amplified with the universal primers TAReuk454FWD1 (5-CCAGCASCYGCGGTAATTCC-3) and 8 (5-ACTTTCGTTCTTGATYRA-3) (5). The PCR program included an initial denaturation at 95C for 5 min, followed by 35 cycles of denaturation at 95C for 30 s, annealing at 56C for 15 s, and elongation at 72C for 1 min, and the last extension step at 72C for 10 min. Due to low DNA quantities in some cases, PCR amplifications for these samples were executed using a nested PCR approach: the primers Euk-A and Euk-B (5-ACCTGGTTGATCCTGCCAG-3, 5-TGATCCTTCYGCAGGTTCAC-3) (25) were applied in the first round PCR as follows: initial denaturation at 94C for 5 min, followed by 20 cycles of denaturation at 94C for 30 s, annealing at 58C for 30 s, and extension at 72C for 30 s, and with the last extension step at 72C for 8 min. One l of product was then amplified in the second round of PCR with the primers TAReuk454FWD1 and TAReukREV12 as described above.