Nucleomag rna kit

Nucleic acids were extracted from filter samples using the NucleoMag plant kit (Macherey-Nagel) for genomic DNA (gDNA) and the NucleoMag RNA kit (Macherey-Nagel) for RNA. Initial sample lysis buffer resuspension and vortexing was completed manually, the remainder using an epMotion liquid handling system (Eppendorf). gDNA was quantified using the Quant-iT PicoGreen double-stranded DNA assay kit (Invitrogen), and RNA was quantified using the Quant-iT RiboGreen RNA assay kit. Nucleic acid integrity was confirmed using an Agilent 2200 TapeStation (Agilent). RNA was reverse-transcribed into cDNA using the SuperScript III first-strand cDNA synthesis system (Invitrogen). Genomic DNA was extracted from Vibrio-enriched pellets using a DNeasy blood and tissue kit (Qiagen), with subsequent quantification and quality control as described above.

32 fibroblast cell lines, including those with NOD (n = 6), NZO (n = 6), and NOD/NZO heterozygous (n = 4) haplotypes at Chr10:82.89 Mbp (GRCm38) were thawed into 60-mm cell culture-treated plates and grown to confluency (≥ 0.8 x 10⁶ cells/ml) in fibroblast media. Each cell line was then passaged equally into two 60-mm cell culture dishes and grown to 75% confluency, upon which one 60-mm dish received 0.75 μM MMA^III containing fibroblast media and one 60-mm dish received standard fibroblast media. Following 24-hr exposure, both treated and untreated samples were independently collected as cell pellets and snap frozen on dry ice for 15 minutes. Samples were stored at -80°C prior to RNA isolation. RNA was extracted using a NucleoMag RNA Kit (Macherey Nagel) and purified with a KingFisher Flex system (ThermoFisher). Library preparation was enriched for polyA containing mRNA using the KAPA mRNA HyperPrep Kit (Rocher Sequencing and Life Science). Paired-end sequencing was performed with a read-length of 150 bp on an Illumina NovaSeq 6000. Genes were tagged as “not expressed” and filtered out if the the median TPM was < .05 for half or more of the samples and the remaining expressed genes were highlighted in “gold” in the variant association mapping plots.

RNA isolation was performed using a KingFisher Flex Purification System and a NucleoMag VET Kit (Macherey-Nagel, no. 744200.4) for the serum and swab samples as well as a NucleoMag RNA Kit (Macherey-Nagel, no. 744350.4) for the tissue samples, respectively, following the manufacturer’s instructions. 10 μL (2 × 10⁴ copies/μL) of enhanced green fluorescent protein (eGFP) RNA were added to each RNA isolation reaction as an internal control to verify the performance of the RNA isolation and real-time RT-PCR, as previously described (67 (link)), with modified primers and probe (Table S4).