16S rRNA amplicon sequencing of gut microbiota of rats
The variable V4 region of the 16S rDNA gene was amplified using the primer pair 515F-Y (5-GTGYCAGCMGCCGCGGTAA-3) and 806R (5-GGACTACNVGGGTWTCTAAT-3). Polymerase Chain Reactions were performed in triplicate (1020 ng of DNA) with a 2x Kapa HiFi HotStart Ready PCR kit and a final primer concentration of 0.25 M in a final reaction volume of 25 L, following the PCR conditions: initial denaturing at 95 C for 3 min, 35 cycles each at 98 C for 20 s, 55C for 30 s, and 72 C for 30 s, followed by a final extension step at 72 C for 5 min. The PCR products were visually assessed via 2 % agarose gel electrophoreses and then purified with Agencourt AMPure XP beads. The libraries were prepared with the Nextera XT indexes (Illumina, USA). Amplicon PCRs for each sample were quantified on the Qubit (Quant-IT dsDNA Broad Range Assay Kit, Invitrogen, Carlsbad, CA, USA), pooled in equimolar ratios and paired-end sequenced of 150 bp on the Illumina MiSeq platform using v2 chemistry according to the manufacturers instructions.
SUB10027145
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