Bacterial 16S rRNA gene amplification

The V1-V2 region of the bacterial 16S rRNA gene was amplified by polymerase chain reaction (PCR) using the universal primers 27Fmod (5′-AGRGTTTGATYMTGGCTCAG-3′) and 338R (5′-TGCTGCCTCCCGTAGGAGT-3′) as described previously^{33 (link)}. Amplicons generated from each sample (~ 330 bp) were subsequently purified using AMPure XP (Beckman Coulter). DNA was quantified using a Quant-iT Picogreen dsDNA assay kit (Invitrogen) and a TBS-380 Mini-Fluorometer (Turner Biosystems). The 16S rRNA gene sequencing was performed with the MiSeq sequencing system (Illumina, San Diego, CA, USA) according to Illumina protocol.

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Masuoka H., Suda W., Tomitsuka E., Shindo C., Takayasu L., Horwood P., Greenhill A.R., Hattori M., Umezaki M, & Hirayama K. (2020). The influences of low protein diet on the intestinal microbiota of mice. Scientific Reports, 10, 17077.

Publication 2020

AMPure XP Quant-iT Picogreen dsDNA assay kit TBS-380 Mini-Fluorometer MiSeq sequencing system

16s rrna Assay Bacterial gene Dsdna Gene Picogreen Polymerase chain reaction Primers

Corresponding Organization : RIKEN Center for Integrative Medical Sciences

Other organizations : Niigata University of Pharmacy and Applied Life Sciences, Papua New Guinea Institute of Medical Research, James Cook University, Federation University, Waseda University

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Variable analysis

independent variables

Amplicons generated from each sample (~ 330 bp) were subsequently purified using AMPure XP (Beckman Coulter)

dependent variables

DNA was quantified using a Quant-iT Picogreen dsDNA assay kit (Invitrogen) and a TBS-380 Mini-Fluorometer (Turner Biosystems)
The 16S rRNA gene sequencing was performed with the MiSeq sequencing system (Illumina, San Diego, CA, USA) according to Illumina protocol

control variables

The V1-V2 region of the bacterial 16S rRNA gene was amplified by polymerase chain reaction (PCR) using the universal primers 27Fmod (5′-AGRGTTTGATYMTGGCTCAG-3′) and 338R (5′-TGCTGCCTCCCGTAGGAGT-3′) as described previously^{33 (link)}

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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