Nanophotometer n60 n50

Genomic DNA in samples was extracted using GF-1 Soil Sample DNA Extraction Kit (Vivantis, Selangor, Malaysia). The extraction and subsequent analysis was done in triplicates following the manufacturer’s instructions. The quality of the purified DNA was first monitored on 1% TAE agarose gel. The concentration of DNA was measured using spectrophotometer (Implen NanoPhotometer^® N60/N50, Munich, Germany) and fluorometric quantification using iQuant^™ Broad Range dsDNA Quantification Kit (Rockville, MD, USA). These protocols were done as previously described by Sinclair et al. [20 (link)]. Aliquots of 1 μL Genomic DNA (gDNA) were run on 1% TAE agarose gel at 100 V for 60 min.

A total of 400 mg of each faecal sample were used to extract bacterial genomic DNA using innuPREP Stool DNA Kit (Analytik Jena, Germany), following the manufacturer’s protocol (Osman et al. 2020 (link), Abdul-Latiff and Md-Zain 2021 (link)). The presence of purified DNAs was first visualised on a 1% Tris-acetate-EDTA (TAE) agarose gel electrophoresis. The concentration quality of DNA was measured using a spectrophotometer (Implen NanoPhotometer® N60/N50), while fluorometric quantification was performed using an iQuant™ Broad Range dsDNA Quantification Kit.

At the 72-hour post-transfection time point, total RNA was extracted using cold Trizol (Invitrogen, USA) and absolute ethanol (Merck, Germany). Subsequently, RNA was isolated and purified using the Direct-zol RNA Miniprep Kit (Zymo Research, USA). The quality and quantity of RNA were assessed using a nanodrop spectrophotometer (NanoPhotometer N60/N50, Implen GmbH, Germany) at 260/280 nm. First-strand cDNA was synthesized from 1 μg of total RNA using the iScript cDNA Synthesis Kit (Bio-Rad, USA) following the manufacturer’s instructions. The resulting cDNA was stored at -20°C until needed for use in RT-qPCR.

Total RNA was isolated from wild-type or knockout cell lines using a PureLink RNA Mini Kit (Invitrogen, Carlsbad, CA, USA), in accordance with the instructions of the manufacturer. The quantity and quality of isolated RNAs were analyzed by UV spectrophotometry (NanoPhotometer N60/N50; Implen, München, Germany). cDNA was synthesized from total RNA using SuperScript III first-strand synthesis supermix (Invitrogen, Carlsbad, CA, USA), and qPCR was performed on a LightCycler 96 system (Roche, Mannheim, Germany) using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA). Gene-specific primers used for qPCR are listed in Supplementary Table S5. Expression levels of genes were normalized to those of glyceraldehyde-3-phosphate dehydrogenase (Gapdh).

Total RNA isolation was performed by commercially available RNA isolation kit from Zymoresearch according to the manufacturer’s protocol. Direct-zol miniprep kit was used for RNA isolation from tissues such as the liver and the adipose tissue, whereas the Direct-zol microprep kit was used for RNA isolation from cultured cells. The quality (A₂₆₀/A₂₈₀) and the quantity (ng/µL) of the RNA were measured by NanoPhotometer N60/N50 (Implen). A ratio of ~2 for A₂₆₀/A₂₈₀ was accepted as good quality RNA.

RNA sequencing (RNA-seq) was performed on lysed frozen PBMCs from 48 randomly selected intervention study participants (19 in the placebo group, 10 in the low dose group, and 19 in the high dose group). These individuals were the same as those subjected to ex vivo PBMC analysis. Each participant had paired samples taken at baseline and on day 14 of the research prior to infection with diarrheagenic E. coli, totaling 96 samples. On the study day, the PBMCs were lysed using buffer RLT (Qiagen 79216, Germantown, MD, USA) and stored frozen until the RNA extraction day. On that day, the samples were thawed, and the total RNA from the cells was extracted according to the manufacturer’s procedure using the RNeasy mini kit (Qiagen 74106, Germantown, MD, USA). Following that, Implen NanoPhotometer N60/N50 was used to quantify the extracted RNA, and the quality and integrity of the RNA samples were checked using the Agilent 2200 TapeStation system (Agilent, Santa Clara, CA, USA) according to the manufacturer’s protocol. RNA samples with an RNA integrity number (RIN) of 8 were used for library preparation. Library construction and sequencing were performed by Novogene (Milton Road, Cambridge, UK), where 2 × 150 bp RNA-seq reads were obtained using Illumina sequencing using a strand-aware library preparation technique.

The mRNA levels of nitric oxide synthase 2 (NOS2), arginase 1 (ARG1), transforming growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α), interleukin-6 (IL6), interleukin-10 (IL-10), chemokine (C-X-C motif) ligand 2 (CXCL2), cluster of differentiation 86 (CD86) and cluster of differentiation 206 (CD206) were detected by real-time quantitative PCR (RT-qPCR). Firstly, RAW264.7 cells were exposed to 0 or 50 μM AFB1 for 24 h. After treatment, cells were washed with PBS and harvested into 1 mL TRIzol (Invitrogen, Waltham, MA, USA) and total RNAs were extracted according to the manufacturer’s instructions. The quantities and qualities of RNAs were evaluated using a NanoPhotometer® N60/N50 (Implen, München, Germany). The complementary DNA (cDNA) was reverse-transcribed from 10 ng of total RNA using a ReverTra Ace^® qPCR RT Master Mix with gDNA Remover Kit (TOYOBO, Osaka, Japan). The levels of the transcripts of the target genes were determined using SYBR^® Green Realtime PCR Master Mix Kit (TOYOBO, Osaka, Japan) on an RT-qPCR system (BIO-RAD, Hercules, CA, USA). The relative amount of target mRNA was performed using the −2^−ΔΔCt method with GAPDH as a reference gene. The primers used in RT-qPCR reactions were synthesized by Sangon Biotech (Shanghai, China) and are showed in Table 3.