Simplinano spectrophotometer

Fecal samples were later thawed and approximately 100-200 mg of fecal sample was used to isolate DNA using spin column chromatography-based Stool DNA Isolation Kit (Norgen Biotek Corp; Product #27,600) following manufacturer recommendations. DNA was quantified with a GE SimpliNano spectrophotometer and shipped on dry ice to LC Sciences, LLC (Houston, TX, USA) for 16S rRNA sequencing. A library was generated by amplifying the V3 and V4 16S rRNA variable regions and adding sequencing adapters and barcodes after the first cycle. An Illumina cBot system was used to generate clusters for sequencing with an Illumina MiSeq platform. Barcodes were used to separate data, and an in-house script was used to annotate taxa according to RDP, Greengenes, and NCBI 16SMicrobial customized databases for reference. Data output statistics, including clustering into operational taxonomic units (OTU), diversity analysis, species classification, and abundance analysis were performed by LC Sciences, LLC (Houston, TX). FASTQ files were deposited in the NCBI Sequence Read Archive with the BioProject accession number PRJNA870961.

Total RNA was isolated from cells using TRIzol (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. Samples were further purified with the Direct-zol RNA Miniprep Kit (Zymo Research). RNA was quantified using the Simpli-Nano Spectrophotometer (GE, Boston, MA, USA).

Astrocytes and pericytes were harvested at the designated time points (Fig. 1). RNA was isolated from the cells using QIAGEN RNeasy Plus Kits (cat #74034) as described previously [33 ], the messenger RNA (mRNA) was quantified using SimpliNano spectrophotometer (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA) and stored in a − 80°C deep freezer for downstream analysis. 200 ng of RNA from each sample were converted into cDNA using SuperScript^™ III First-Strand Synthesis SuperMix (catalog # 11752-050; ThermoFisher SCIENTIFIC) as per manufacturer’s instructions. The Quantitative RT-PCR assays were performed using TaqMan^® Gene Expression Assay (20 ) and TaqMan^™ Universal PCR Master Mix (Catalog number: 4304437). The thermal cycling conditions were set to initial denaturation 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec, 60°C for 1 min. The primer/probe set used in the study was as follows: GAPDH as endogenous control (Hs02758991_g1; VIC/MGB), ACE2 (Hs01085331_m1; FAM/MGB), TMPRSS2 (Hs00237175_m1; FAM/MGB), NRP1 (Hs00826128_m1; FAM/MGB), TRIM28 (Hs00232212_m1; FAM/MGB), IL-6 (Hs00174131_m1; FAM/MGB), TNF-α (Hs00174128_m1; FAM/MGB), IL-1β (Hs01555410_m1; FAM/MGB) and IL-18 (Hs01038788_m1; FAM/MGB). The fold change in gene expression values was calculated using the 2^−Δ(ΔCt) method; where ΔCt = Cttarget—Cthousekeeping and Δ(ΔCT) = ΔCt infected - ΔCt mock-infected.

Global 5mC levels in tomato genomic DNA was determined as previously described with minor modifications [68 (link)]. In brief, DNA was extracted from the pericarp tissues using Sureplant DNA kit (Cwbiotech, CW2298), with the disruption of total RNA according to the manufacturer’s protocols. The extracted DNA was detected in 1% agarose gel and quantified by a SimpliNano spectrophotometer (GE Healthcare, 29-0617-11). Then, 100 ng of purified and integrated DNA for each measurement was used to perform 5mC assay by MethylFlash™ methylated DNA quantification kit (Epigentek, P-1034). 5mC levels in different DNA samples were relatively quantified using both the negative control and positive control, which contain 0% 5mC and 50% 5mC, respectively, following the manufacturer’s instructions.