Primer blast

Primers targeting a highly conserved gene and an intergenic region were designed for the detection of S. Typhi and S. Paratyphi A serovars respectively (Table 2). Primers with additional mutations incorporated based on the mismatch amplification mutation assay (MAMA) PCR principle as previously described [18 (link)], were designed to selectively amplify a target sequence in the presence of a SNP of interest for H58 and XDR lineages [19 ]. All primers were designed using Primer-BLAST [20 (link)], synthesised by Integrated DNA Technologies (IDT, USA) and were resuspended from their lyophilised form in 10 mM Tris buffer. Appropriate amplicon lengths were selected for each target with a minimum of 50 base pairs (bp) difference, in order to effectively separate and be visualised by gel electrophoresis.

TEP sequences identified in the BB02 reference genome were used as template to design PCR, qRT-PCR, and 5’- and 3’-RACE primers with the National Center for Biotechnology Information’s (NCBI) Primer-BLAST and Integrated DNA Technologies (IDT) PrimerQuest online tools. Primers were purchased from the Custom DNA Oligo Synthesis service (Eurofins Genomics) (Supplemental Table 1). cDNA samples from each snail strain were then used to amplify each TEP using PCR and GoTaq G2 Green Master Mix (Promega). In addition, the Q5 High-Fidelity 2X Master Mix (New England Biolabs) was also used to verify the fidelity of the original sequences using several PCR primer sets (Supplemental Tables 1–3). Primers designed for RACE (Supplemental Table 4) were used to extend the unknown 5’ and 3’ ends of incomplete sequences using the GeneRacer kit (Thermo Fisher Scientific). Transcripts of interest included 11 members of the TEP superfamily identified using the BB02 reference genome (A2M-1, A2M-2, CPAMD8, C3-1, C3-2, C3-3, TEP-1, TEP-2, TEP-3, TEP-4, and CD109) as reported in Castillo et al. (10 (link)). As a positive control, the housekeeping gene, ribosomal protein subunit 19 (RPS19) was also amplified.

Specific primers for qRT-PCR were designed using Primer-BLAST (65) and OligoAnalyzer 3.1 software (Integrated DNA Technologies) (Zhu et al., 2018 (link)). Then, qRT-PCR was performed using SYBR Green PCR Master Mix (TaKaRa) on a Real-Time PCR System (Rotor-Gene Q MDx, Germany). The levels of miRNAs and TPTEP1 were normalized to those of the internal control of the kit and RNU6 (U6), respectively (Le Cras et al., 2020 (link)). GAPDH was used as an internal control to normalize the expression of other mRNAs. Relative gene expression was calculated using the 2^–ΔΔCt method (Zhang F. et al., 2020 (link)).

Both cell lines were cultured in a 6-well plate at a density of 4 × 10⁵ cells/mL, and after treatment with the adipocyte secretome, the RNA was extracted using the same extraction system and method that was also used in Section 2.3. The inflammatory state was then assessed via the expression of inflammatory genes (IL-1β, IL-4, IL-6, and IL-10) using gene-specific oligonucleotide primers (Table 2) that were designed using PrimerBlast and purchased from Integrated DNA Technologies, Europe. RT-qPCR detection was conducted using the One-Step NZYSpeedy RT-qPCR Green Master Mix (NZYTech), under the following cycle conditions: incubation at 50 °C for 20 min (reverse transcriptase action), 95 °C for 3 min (polymerase activation), and 40 cycles of 95 °C/10 s and 60 °C/40 s. The relative quantification was determined as a -fold increase of the gene of interest compared with the 18S housekeeping gene (reference gene) using the 2^−ΔΔCt method. The assay was conducted in duplicate.

Frozen hippocampal tissue was homogenized with a hand-held homogenizer (UX-44468-25, Cole-Parmer) for 15 s and total RNA was isolated from the homogenate using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. qPCR was performed as previously described^{33 (link)}. Primers obtained from Integrated DNA Technologies were designed to span exon-exon junctions using Primer-BLAST. Primer sequences and efficiencies are shown in Supplemental Table 1.