title: 16SrRNA-Seq of bacteria
design: Three grams of sand sample was taken from the first layer (~ 2 cm) of each drained column and stored in sterilized centrifugal tube at -80C until extraction. DNA was extracted using a FastDNA spin kit for soil (Bio101 Inc., Melbourne, Australia) according to the manufacturer's instructions. The quality and concentration of DNA were determined by 1.0% agarose gel electrophoresis and a NanoDrop2000 spectrophotometer (Thermo Scientific, USA) and kept at -80 before further use.The hypervariable region V3-V4 of the bacterial 16S rRNA gene were amplified with primer pairs 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') using a thermocycler PCR system (GeneAmp 9700 Applied Biosystems, Foster City, CA, USA). The PCR reaction was performed according our previous study (Xia et al., 2020).Purified amplicons were pooled in equimolar concentration and paired-end reads were sequenced (2  300) on an Illumina MiSeq platform (Illumina, San Diego, USA) according to the standard protocols of Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China).