RNA-Seq of Sea bulk water 20 m - Transect St. 2
DNA were extracted using the DNeasy PowerSoil Pro Kit (Qiagen) according to the manufacturers protocol (HB-2495-002  2018, Qiagen). Amplification of the 16S rRNA gene was performed using specific primers. The hypervariable V3 and V4 regions of the 16S rRNA gene were amplified with the primers Bac341F (5-CCT ACG GGN GGC WGC AG-3) and Bac805R (5-GAC TAC HVG GGT ATC TAA TCC-3). The amplification process followed the Illumina protocol (16S Metagenomic Sequencing Library Preparation; part # 15044223 Rev B), with slight modifications depending on the sample type.For the 16S rRNA amplification, the first PCR-mixture contained 11 L template DNA for air and plant samples and 3 L for seawater and soil samples, 12.5 L KAPA HiFi HotStart polymerase (Kapa Biosystems, Inc., Wilmington, MA, United States), 0.5 L forward primer (10 pmol/L), 0.5 L reverse primer (10 pmol/L), 0.5 L bovine serum albumin (BSA) (4 g/L), and finally added up with ddH2O to a total volume of 25 L per reaction. Thermal cycling included an initial denaturation step at 90C for 3 min, followed by 22 cycles for seawater and soil samples, otherwise 30 cycles, of denaturation at 95C for 30 s, annealing at 55C for 30 s, elongation at 72C for 30 s, and a final elongation at 72C for 5 min. PCR products were purified using 30 L AMPure XP magnetic beads. In the second PCR, Illumina overhang adaptors were added. The reaction mixture contained 2 L template DNA for plant and soil samples, and 3 L template DNA for seawater and air samples, 12.5 L KAPA HiFi HotStart polymerase, 0.5 L forward primer + adaptor (10 pmol/L), 0.5 L reverse primer + adaptor (10 pmol/L), and finally added up with ddH2O to a total volume of 25 L per reaction. No BSA was added. This second PCR was run for 15 cycles for the air samples, while the rest of the samples were run for 10 cycles under the same thermal cycling conditions as for the first PCR. Following the second PCR, the resulting samples were quality-controlled by gel electrophoresis on a 1.5% agarose gel. PCR products were purified with 20 L AMPure XP magnetic beads. The third PCR incorporated Nextera XT Index primers. The reaction mixture contained 3 L template DNA for the seawater and air samples and 2 L template DNA for the plant and soil samples, 12.5 L KAPA HiFi HotStart polymerase, 2.5 L of each index primer (N7XX and S5XX), and added up with ddH2O to a total volume of 25 L per reaction. Thermal cycling was performed for eight cycles for all samples under the same conditions as for the first two PCR, and PCR products were purified using 20 L AMPure XP magnetic beads. The concentration of the final PCR products was measured using the Quant-iTTM dsDNA BR assay kit on a FLUOstar Omega fluorometric microplate reader (BMG LABTECH, Ortenberg, Germany). The PCR products were then diluted and pooled in an equimolar ratio. The pooled library was quantified using the Quant-iTTM dsDNA BR assay kit on a Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, United States) and sequenced on the Illumina MiSeq platform (Illumina, San Diego, CA, United States), generating 2x300-bp long paired-end reads.