Aati fragment analyzer

The analysis of gene expression in the immune response was performed using the NanoString nCounter™ Mouse Inflammation v2 Panel (NanoString Technologies, Inc., Seattle, WA, USA). For sample preparation, total RNA was extracted from RAW264.7 cells after treatment. The quantity and quality of the extracted RNA were measured using a DS-11 spectrophotometer (DeNovix Inc., Wilmington, DE, USA) and an AATI Fragment Analyzer (Agilent Technologies, Santa Clara, CA, USA). The samples with sufficient RNA purity, demonstrated by an RNA concentration of 20 ng/μL or more and an acceptable 260 nm/280 nm absorbance ratio were used. Total RNA was hybridized to a reporter-capture probe at 65 °C for 24 h. After the hybridization reaction, the samples were loaded onto an nCounter cartridge (NCT-120) and the data were gathered by the nCounter Prep Station and the nCounter Digital Analyzer. The raw data were normalized to the housekeeping gene expression and expressed as fold-change. A heatmap was generated using the normalized data.

Plasmid characterization was performed by PBRT [10 (link)] using the PBRT kit 2.0 (Diatheva, Fano, Italy). This system, consisting of eight multiplex PCR assays, allows the identification of the following 30 replicons found in the Enterobacteriaceae family: HI1, HI2, I1, I2, X1, X2, X3, X4, L, M, N, FIA, FIB, FIC, FII, FIIS, FIIK, FIB KN, FIB KQ, W, Y, P1, A/C, T, K, U, R, B/O, HIB-M and FIB-M. All PCR reactions were performed according to the manufacturer’s instructions, including positive controls. The amplicons were detected through capillary electrophoresis on the AATI Fragment Analyzer (Agilent, Santa Clara, CA, USA) using the dsDNA 906 Reagent kit (Advanced Analytical, Ankeny, IA, USA). This amplicon analysis allows the combination of two multiplex PCRs in the same lane, resolving up to eight peaks. One µL of multiplex PCR 1 (M1) was combined with 1 µL of multiplex PCR 3 (M3), followed by M2 and M7, M6 and M8; the remaining M4 and M5 were loaded separately (2 µL each). The positive peaks were analyzed using the “PBRT plugin” developed in cooperation with the Advanced Analytical Company. This tool allows automatic peak calling and the recording of positive replicons.

Bacterial DNA was obtained by the boiling lysis method, incubating the isolated colonies in distilled water for 10 min at 100 °C. The samples were then centrifuged at 15,000× g for 5 min and the supernatants were used for the following reactions.
All strains were typed by PCR-based replicon typing (PBRT) using the PBRT kit 2.0 (Diatheva, Fano, Italy) in order to identify plasmid replicons. This PBRT assay consists of eight multiplex PCRs and allows the detection of 30 replicons of the main plasmids in Enterobacterales.
All PCR reactions were carried out in accordance with the manufacturer’s instructions, including positive controls. The amplicons were detected through capillary electrophoresis on the AATI Fragment Analyzer (Agilent, Santa Clara, CA, USA) using the dsDNA 906 Reagent kit (Advanced Analytical, Ankeny, IA, USA). This amplicon analysis allows the combination of two multiplex PCRs in the same lane, resolving up to eight peaks, as previously published [58 (link)]. The positive peaks were successively analysed using the tool “PBRT plugin” [58 (link)] developed in cooperation with the Advanced Analytical Company that allows automatic peak calling and the recording of positive replicons.

The PBRT 2.0 kit (Diatheva, Fano, Italy) which has been used for plasmid identification and to type the major resistance plasmids found in Enterobacteriaceae (Carloni et al., 2017 (link)), was applied in our study. The amplicons recognized by the PBRT kit were analyzed by capillary electrophoresis with an AATI Fragment Analyzer (Agilent, Santa Clara, CA, United States).
Where necessary, amplicons were purified using MinElute PCR purification kit (Qiagen, Hilden, Germany) and directly sequenced by the Sanger method using BigDye Terminator v. 1.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Vilnius, Lithuania) on the ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, United States). The consensus sequences obtained were submitted to the pMLST database for allele variant identification².
The stx1, stx2, and eae genes of shiga toxin-producing E. coli (STEC) strains were identified using STEC FLUO detection kit (Diatheva, Fano, Italy) according to the manufacturer’s instructions; the strains that were positive for stx and/or eae genes underwent O-serogroup identification (O157, O111, O26, O103, and O145) using the STEC Serotypes FLUO kit (Diatheva).

To aim for partitioning of 7,000 cells with the expectation that 3,500 cells would be sequenced, 6 µL of the protoplast suspension with an estimated 1,200 cells/µL was applied to the 10× genomics chromium microfluidic chip (Chemistry V3.0). Thereafter the standard manufacturer’s protocol was followed. Twelve cycles were used for cDNA amplification, and the completed cDNA library was quantified using an Agilent AATI Fragment Analyzer. Sequencing was performed at Novogene (Sacramento, CA, USA) on a single lane with the Hi-Seq platform and the standard PE150 sequencing parameters.

The nCounter SPRINT platform (NanoString Technologies, Inc. Seattle, WA, USA) was used to analyze the gene expression by microarray, as described previously [21 (link)]. Briefly, RAW 264.7 cells were seeded on 6-well plates at a density of 4 × 10⁵ cells/well, and incubated for 24 h. The cells were treated with CSP32 and l ng/mL LPS for 24 h, and then, the total RNA was isolated by TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. To evaluate for RNA condition, all samples were performed quality control test and quantitative analysis using AATI fragment analyzer (Agilent Technologies, Santa Clara, CA, USA) and DS-11 spectrophotometer (DeNovix Inc., Wilmington, DE, USA). After solution-phase hybridization between the target mRNA and reporter-capture probe pairs, the excess probe was removed, and the probe/target complexes were aligned and immobilized in the nCounter cartridge (NCT-120), which was then placed in a digital analyzer for image acquisition and data processing. The raw data were normalized with 14 housekeeping genes, including ALAS1, EEF1G, G6PDX, GAPDH, GUSB, HPRT, OAZ1, POLR1B, POLR2A, PPIA, RPI19, SDHA, TBP, and TUBB5. Fold change data was log2-transformed and log2 fold-change cut-offs of −2.5 and 2.5 (red and green, respectively) in the expression of the selected genes are presented in a heat map.