RG2c replicate 2
Paddy soil samples were collected at depth of 0-5 cm, 15-20 cm, and 35-40 cm in three replicates, dried in frozen state, and stored at -20C. Total DNA was extracted from about 250 mg dried paddy soil using Power Soil DNA isolation kit (Qiagen GmbH, Germany). PCR was carried out to amplify the v4 regions of 16S rRNA genes of bacteria and archaea as previously (Nguyen et al. 2015). The primers are detailed as follows: forward primer (AATGATACGGCGACCACCGAGATCTACAC-XXXXXXXX-TATGGTAATT-GT-GTGCCAGCMGCCGCGGTAA) and reverse primer (CAAGCAGAAGACGGCATACGAGAT-XXXXXXXX-AGTCAGTCAG-CC-GGACTACHVGGGTWTCTAAT) where X denotes nucleotides in specific sequences used to index samples.Two microliters of the total DNA from each sample was used as a PCR template,and amplification was done in triplicates using iTaq mastermix 2X (iNtRON Biotechnology Inc., Korea) with the following conditions: 95C for 2 min; 33 cycles of 95C for 20 s, 55C for 15 s, and 72C for 5 min; and 72C for 10 min. Obtained PCR products were further gel-purified using Qiaquick gel extraction Kit (Qiagen GmbH, Germany). All obtained DNAs were quantified and equimolar purified amplicons were pooled and stored at ?20C until use. Illumina MiSeq platforms were used to sequence the v4 regions of the 16S rRNA genes (3002) by Macrogen Inc. (Seoul, Republic of Korea).