16S rRNA amplicons of Anthonomus grandis gut microbiome: fed with corn stover
DNA was extracted from gut samples using the DNeasy Blood and Tissue kit (Qiagen, USA) according to the manufacter쳒 instruction. The V3-V4 hypervariable regions of 16S rRNA gene were amplified in triplicate from microbial genomic DNA using the following universal primers: 515F (5_-GTGCCAGCMGCCGCGGTAA-3) and 806R (5_-GGACTACHVGGGTWTCTAAT-3_) with sequencer adapters and sample-specific Golay barcodes on the reverse primer. Polymerase chain reaction (PCR) were performed in triplicate per sample by using 1 _ Takara ExTaq PCR buffer with MgCl2, 10 _M of primers, 0.56 _g/킠 bovine serum albumin, 200__M dNTPs, 0.025_U ExTaq DNA polymerase (Takara Mirus Bio Inc., Madison, WI, USA), 10_ng template and milliQ H2O to complete 25__L volume. PCR cycling was performed with an initial denaturation at 95좧 for 3_min, followed by 30 cycles at 95좧 for 30_s, annealing at 55좧, for 45_s, extension at 72좧 for 90 s and a final extension of 72좧 for 12_min. The triplicate reactions were pooled and purified using SPRI beads (Agencort AMPure XP, Beckam Coulter Inc. Brea, CA, USA) magnetic beads (1.2 x _L of sample volume) to select for 400 bp amplicons according to the manufacturer쳒 protocol. The samples were then quantified using a Qubit fluorometer (Qiagen). Concentrations of each sample were calculated and then diluted to 10 nM. All samples were pooled in equimolar amounts for sequencing. The amplicons were sequenced using the Illumina MiSeq kit v3 and 300 PE sequencing cycles.