OEX011312
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OEX_xiafei_2101091650 |
Metagenomic |
Extraction and purification of protist community DNA from filtered water and sediment were carried out using the PowerWater DNA Isolation Kit and PowerSoil DNA Isolation Kit (MO BIO laboratories, Carlsbad, USA), respectively. For sediments, we combined the freeze-grind DNA extraction method with the MoBio PowerSoil kit as described in http://www.ou.edu/ieg/tools/protocols. The quantity and quality of DNA were determined using a NanoDrop Spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA). The V4 region of the eukaryotic small-subunit 18S rRNA gene was amplified using the universal primer pair TAReuk454FWD1 (5′-CCAGCA(G/C)C(C/T)GCGGTAATTCC-3′) and TAReukREV3 (5′-ACTTTCGTTCTTGAT(C/T)(A/G)A-3′) (Stoeck et al., 2010). The PCR program was composed of an initial denaturation step at 95 °C for 5 min; 30 cycles of 94 °C for 30 s, 47 °C for 45 s and 72 °C for 1 min; and a final 5-min extension at 72 °C (Yang et al., 2018). The PCR product of each sample was purified with equal quantity and sequenced on the Illumina HiSeq2500 platform (Illumina, Inc., San Diego, CA, USA) using a paired-end (2 × 250 bp) approach. |
2021-01-09 |