Basespace cloud computing platform

The library preparations for next-generation sequencing and Illumina MiSeq sequencing were conducted by GENEWIZ, Inc. (South Plainfield, NJ). The DNA samples were quantified using a Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA) and DNA quality was confirmed by electrophoresis (0.8 % agarose gel). The sequencing library was constructed using a MetaVx™ Library Preparation kit (GENEWIZ, Inc., South Plainfield, NJ). In brief, 5–50 ng of DNA was used for amplicon generation to cover the V3 and V4 hypervariable regions of 16S rDNA. Indexed and universal adapters were added to the ends of the 16S rDNA amplicons by limited-cycle PCR. The DNA libraries were validated with an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) and quantified using Qubit and real-time PCR (Applied Biosystems, Carlsbad, CA). The DNA libraries were multiplexed and loaded on an Illumina MiSeq following the instructions from the manufacturer (Illumina, San Diego, CA). A 2 × 150 paired-end (PE) configuration was used for sequencing. The image analysis and base calling were processed using MiSeq Control Software. The initial taxonomy was performed on Illumina’s BaseSpace cloud computing platform.

Caecal contents samples were collected at 8 weeks from rats from different groups, using a QIAamp-DNA stool mini kit (Qiagen, Hilden, Germany) to extract Metagenomic DNA from caecal contents of rats. The V3-V4 hypervariable regions of 16S rRNA gene from caecal microbiota were amplified using specific primers (F: 5′-CCTACGGRRBGCASCAGKVRVGAAT-3′ and R: 5′-GGACTACNVGGGTWTCTAATCC-3′) [80 (link)]. Sequencing was performed using a 2 × 300 paired-end (PE) configuration. Analysis was performed by MiSeq control software. The initial classification analysis was conducted on an Illumina’s Base Space cloud computing platform.

Samples were pooled into single library using TruSeq Stranded mRNA Library Prep and sequenced with the NextSeq 500/550 Mid Output Kit v2.5 (150 cycles) on the Illumina NextSeq 500 platform (Illumina, San Diego, CA, USA). Sequenced reads were assessed for quality using the Illumina Basespace Cloud Computing Platform and FASTQ sequence files were used to align reads to the mouse reference genome [Mus musculus/UCSC mm9] using RNA-Seq Alignment Application with STAR aligner. Fragments per kilobase of transcript per million mapped reads (FPKM) values of reference genes and transcripts were generated using Cufflinks 2. Differential expression was determined by univariate analysis and a full list of differentially regulated genes (DRG; P < 0.01) is provided in Dataset S1. The generation of adult rat ventricular myocytes (ARVMs) overexpressing NR4A2 and RNA-Seq on those cells has previously been reported [3 (link)]. Molecular pathways differentially expressed between groups (P < 0.05) were identified and visualized using Reactome v76 (www.reactome.org).

Total metagenomic DNA was extracted from cecal contents of rats using a QIAamp-DNA stool mini kit (Qiagen, Hilden, Germany). The V3-V4 hypervariable domain of 16S rRNA gene was amplified with the special primers (F: 5′-CCTACGGRRBGCASCAGKVRVGAAT-3′ and R: 5′-GGACTACNVGGGTWTCTAATCC-3′). Sequencing was performed using a 2 × 300 paired-end configuration by IonS5^TMXL platform. Phenotypic analysis was carried out by MiSeq control software. The initial classification analysis was conducted on Illumina’s Base Space cloud computing platform.