Soil fungal microbiome from 1975 Oil Spill Site Rep 3
DNA was extracted from 0.5 g of soil using the Exgene soil DNA minikit from GeneAll (Seoul, Korea). The extraction process involved the use of specific buffers, including 550 L of buffer SL (extraction buffer), 50 L of buffer RH, 300 L of buffer PD, 900 L of buffer TB (tissue binding), 500 L of buffer NW (wash buffer N), and 50 L of elution buffer. Each extraction step was followed by centrifugation using a 5810R Eppendorf centrifuge. Subsequently, Polymerase Chain Reaction (PCR) amplification was performed to target the fungal ITS1 18S rDNA. The amplification process utilized the CS2-ITS4 F(GCGGTAATTCCAGCTCCAA) and CS1-ITS2 R(AATCCRAGAATTTCACCTCT) primers (Brown et al., 2016). The PCR was conducted on an Applied Biosystems Veriti 96-Well Thermal Cycler. Each PCR reaction mixture (25 L) consisted of 12.5 L HS Taq Mix Red (PCR Biosystems, London, UK), 9.5 L ultrapure water, 1.0 L extracted DNA, 1 L CS1-ITS2 R primer, and 1 L CS2-ITS4 F primer. The PCR cycles included an initial denaturation at 95C for 2 min, followed by 40 cycles at 95C for 15 sec, 50C for 30 sec, 72C for 30 sec, and a final extension step at 72C for 3 min. The resulting PCR products were sent to Hylabs Inc. for further analysis. Hylabs used Fluidigm Access Array primers for Illumina to generate libraries compatible for sequencing on the Miseq. Prior to sequencing, the concentration of the samples was measured using Qubit, and the size was assessed using TapeStation. Finally, the samples were sequenced on the Illumina Miseq platform using a Miseq V2 sequencing kit (500 cycles) to produce 2x250 paired-end reads.