Dna purification kit

The polymorphic gene MELO3C022157 was PCR-amplified from resistant (PI 353814) and susceptible (PI 614596) accession using Phusion^® High-Fidelity DNA Polymerase (New England Biolabs, EVRY Cedex, France). Amplified DNA fragments were purified using a Promega DNA Purification kit (Promega, Madison, WI, USA). Cloning was performed using a TOPO-TA cloning kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. The universal primers M13F and M13RpUC were used to sequence cloned amplicons using an ABI3730XL sequencer (Macrogen Co., Seoul, Korea). Each forward and reverse sequence of resistant and susceptible melon accession was repeated three times to remove all uncertainties. Gene sequences of the resistant and susceptible accessions were aligned using CLUSTALW software (https://www.genome.jp/tools-bin/clustalw) to detect sequence variation.

Bacterial translocation was determined as bacterial load in liver tissue and was quantified by real-time PCR using the 16S rRNA gene consensus sequence as described by Banerjee et al. [47 (link)]. The total load of bacteria in the liver was determined using primer sequences to amplify the highly conserved sequence for a broad species consensus. Livers were removed aseptically and stored at −80 °C until use. Bacterial translocation was quantified by real-time PCR. Briefly, liver tissue was homogenized in a MP FastPrep 24 Instrument (MP biomedicals, Irvine, CA) using Green Bead Lysis kits (Next Advance, Troy, NY). DNA was isolated from liver lysates using a DNA purification kit (Promega, Madison, WI). Bacterial DNA and liver genomic DNA concentration were quantified using Quant-iT™ PicoGreen™ dsDNA Assay Kit (Thermo Fisher Scientific, Waltham, MA). Serially diluted bacterial genomic DNA was used to generate the standard curve. Real-time PCR was performed using PowerUp SYBR green PCR master mix (Applied Biosystems, Foster City, CA) and 16S rRNA gene targeted primers, forward (5′-ACTCCTACGGGAGGCAGCAGT-3′) and reverse (5′-TATTACCGCGGCTGCTGGC-3′) in a QuantStudio 3 Realtime PCR System (Thermo Fisher Scientific, Waltham, MA). PCR-derived bacterial counts were expressed as nanogram bacterial DNA per gram mouse liver tissue.

Genomic DNA was isolated from 5 × 10⁶ cells using the DNA Purification Kit (Promega). Whole-genome sequencing (WGS) was performed and analyzed at the Yale Center for Genome Analysis (West Haven, CT). Using Illumina's Sequence Analysis Viewer, various quality control (QC) parameters were monitored to assess the sample quality. The sequencing reads passing the QC parameter values were aligned to the hs38DH human reference for variant calling. The GATK MuTect2 was used to call and filter somatic variants.

Genomic DNA was extracted using a DNA Purification Kit (Promega). Bisulphite treatment of extracted genomic DNA was performed using the EZ DNA Methylation Kit (Zymo Research). The bisulphite-converted genome was amplified by PCR using bisulphite-specific primers for Zfp423 and ZNF423 (see ESM Table 1 for the primers used). Bisulphite genomic sequencing was performed as previously reported [27 (link)]. DNA sequencing was performed on an ABI 3500 Automatic Sequencer using Big Dye Terminator v3.1 (Applied Biosystems). Bioinformatic analysis was carried out using EMBOSS CpGplot (available from www.ebi.ac.uk/Tools/seqstats/emboss_cpgplot/; accessed November 2015).