Qubit dsdna assay kit

DNA was extracted from ≈200 mg of the compounded root soil using an ultra-clean microbial DNA isolation kit (MO BIO, QIAGEN, Germantown, MD), and extracts were purified using a OneStep PCR inhibitor removal kit (Zymo). DNA was quantified using a Qubit dsDNA assay kit (Invitrogen). Metagenome libraries were prepared using an Illumina DNA prep kit (Illumina, San Diego, CA). Prior to sequencing (Cornell Sequencing Center), library inputs (metagenome, 16S rRNA) were purified and normalized using a SequalPrep normalization plate kit (Applied Biosystems, Waltham, MA).

Microbial genomic DNA was extracted from fecal samples using a FastDNA Spin Kit (MP Biomedical, Irvine, CA, USA). The V3-V4 region of bacterial 16S rRNA was then amplified using a universal primer pair (341F and 806R). The differential detection of Bifidobacterium species was performed using primers designed to target the groEL gene. PCR products were purified and quantified using a Qubit dsDNA Assay Kit (Life Technologies, Invitrogen, Carlsbad, CA, USA). Purified amplicons were pooled and paired-end sequencing was performed on a MiSeq PE300 platform (Illumina, San Diego, CA, USA).
Raw data were processed using the QIIME tools version 2.0 and bioinformatic analyses of the microbiota were performed. α-Diversity was calculated based on species richness and evenness using Chao 1, Shannon, and Simpson indices. β-Diversity was assessed based on the Bray–Curtis distance and was visualized via principal coordinate analysis (PCoA). The differential abundance of microbial taxa was calculated using linear discriminant analysis effect size (LEfSe) and visualized using a taxonomic cladogram tree. Metagenomes of the gut microbiome were computed from 16S rRNA sequences based on Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt), and functional pathways were predicted using the Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology.

Cyclobutane pyrimidine dimers (CPD) were measured using monoclonal antibodies by dot-blot analysis (TDM-2; Cosmo Bio Co., Ltd., Japan). Twelve-day-old plants were treated with UV-B for 1 or 4 h as described above, and samples (0.1 g) were collected after the treatment. 2 μg of the extracted DNA by a modified cetyltrimethylammonium bromide (CTAB) method was then denatured using 0.3 M NaOH for 10 min. Samples were analyzed using a nylon membrane (PerkinElmer Life Sciences, Inc.) in sextuplicate. The membrane was incubated at 80°C for 2 h and blocked with a buffer containing 20 mM Tris–HCl, pH 7.6, 137 mM NaCl (TBS) and 5% (p/v) dried milk for 1 h at room temperature. The membrane was finally washed with TBS and incubated with anti-CPDs antibodies (1:2000 in TBS) overnight at 4°C with agitation. Unbound antibodies were washed away and secondary antibodies conjugated to alkaline phosphatase (1:3000; Bio-Rad) were added. The blot was washed and finally developed by the addition of 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium. Dots were quantified by densitometry using ImageQuant software version 5.2. Total DNA was quantified fluorometrically using the Qubit dsDNA assay kit (Invitrogen).

Young leaf tissue was collected from each of the 300 genotypes in the GWAS panel, and DNA was extracted using a modified cetyltrimethylammonium bromide (CTAB) extraction protocol following Porebski et al. (1997) (link). Quantification of DNA was performed using the Qubit dsDNA assay kit (Invitrogen, Carlsbad, CA) and samples were standardized to 40 ng/µl. Capture-Seq genotyping was performed at RAPiD Genomics (Gainesville, FL) with 35,054 custom biotinylated 120-mer probes distributed across the R. argutus genome. The majority of the probes were designed to target genic regions, including a number of genes implicated in cell wall metabolism in other fruits such as polygalacturonase (PG), pectinmethylesterase (PME), pectin lyase, and expansin. DNA libraries were sequenced with Illumina HiSeq to achieve an average of 5.14 million 150 bp paired-end reads per sample (Supplementary Table 1).

A Fast DNA Spin Kit for Feces (MP Biomedicals, Irvine, CA, USA) was used to extract total DNA from fecal samples. Universal primers (341F: 5′-CCTAYGGGRBGCASCAG-3′ and 806R: 5′-GGACTACNNGGGTATCTAAT-3′) were used to perform PCR amplification of the 16S rRNA V3–V4 region and the samples were barcoded.
The PCR products were separated on a 1.5% agarose gel and purified using the DNA Gel/PCR Purification Miniprep Kit BW-DC3511 (BIOMIGA, San Diego, CA, USA). The purified PCR products were quantified using the Qubit dsDNA Assay Kit (Life Technologies, Invitrogen, Waltham, CA, USA). In the final step, equal amounts of samples were mixed according to the concentration of PCR products, and an Illumina MiSeq PE300 was used for paired-end sequencing.