Ssoadvanced universal sybr green supermix

Gene-specific regions were amplified from cDNA (5–10 ng/μl) with assay primers (200 nmol/L each) and Bio-Rad SsoAdvanced™ Universal SYBR® Green Supermix (10 μl reaction, 95°C for 3 min, followed by 40 cycles of 95°C for 10 s and 60°C for 20 s) on the ABI 7900HT system (Thermo Fisher Scientific). Assay primers were designed with Primer-BLAST software (29 (link)). Assay primer pairs were designed to span a large intron with binding sites on adjacent exons with similar melting points (Tm = 60) and amplicon sizes ranging from 70 to 120 nucleotides. SDS 2.3 software was used for the experimental setup. Assay sequences are listed in Supplemental Table S2.

The qPCR reactions were performed for the six individuals per treatment in duplicate on a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, Madrid, Spain). Each reaction (20 μl) contained 10 μl SsoAdvanced™ Universal SYBR® Green Supermix (Life Technologies, Carlsbad, California, USA), 0.50 μl forward primer, 0.50 μl reverse primer, 2 μl of cDNA sample, and 7 μl of RNase/DNase-free water. The real-time qPCR protocol started with 95°C for 3 min, followed by 40 cycles of 15 s at 95°C followed by 30 s at the specific annealing temperature for each primer pair followed by 30 s at 72°C with a final melt curve step to establish standard melt curve profiles for each amplicon. Relative expression was calculated using CFX Manager 3.0 (Bio-Rad, Madrid, Spain) software, with elongation factor 1 alpha (EF1α) and hypoxanthine-guanine phosphoribosyltransferase (HPRT) as endogenous controls, while basal (pre-stress) samples from control and Trp groups were used for normalizing the relative quantification within each experimental diet group. All samples were run in duplicate with a negative control (no RT enzyme) included to confirm absence of genomic DNA contamination and an additional negative control on each plate containing no template cDNA.

ChIP was performed as previously described by our laboratory [13 (link)–15 (link)]. Sheared chromatin was incubated overnight at 4 °C with validated ChIP-grade antibodies against H3K9me2 (Abcam, Cat. #ab1220) or REST (Millipore, Cat. #17-641). The resulting DNA was quantified using qPCR with SSOAdvanced Universal SYBR Green Supermix (Life Technologies, Cat. #4367659) and primers for promoter and promoter CpG islands at Chat, Trka, Lhx8, and Parvb genes (Table 3). The ΔΔCt method was used to determine fold occupancy relative to CONs and was normalized to the Input DNA fraction.Table 3

List of primer sequences for ChIP analysis.

Primer	Forward	Reverse	Target
Chat Promoter	ACTTGATTGCTGCCTCTCTC	GGGATGGTGGAAGATACAGAAG	Promoter
Chat CpG Promoter	TGCATCTGGAGCTCAAATCGT	GGGGATAGTGGTGACGTTGT	Promoter CpG island
Trka Promoter	CCTCACCGTGCACTTTACCT	AGGGTCTGGAGAGCGTACAT	Promoter
Trka CpG Promoter	TCAAGCAAGGCTCCGAACAG	CACAGGGTGGCGCTAGAAG	Promoter CpG island
Lhx8 Promoter	ATCGGAGGCGGTGTATGTTC	TGGGCCTGGTTCGGATTAAG	Promoter
Parvb Promoter	CAGAAGATGGTCCCTGACGG	ACCTTCAAGCTGAACGGGTC	Promoter

RNA was purified from freshly activated T cell blasts using the RNeasy Plus Mini Kit (Qiagen GMbH, Hilden, Germany). Genomic DNA was removed and the RNA transcribed to cDNA as described [16 (link)]. qPCR was performed using the SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) according to the manufacturer’s instructions, using a 10 µl reaction mixture [5 µl SsoAdvanced Universal SYBR Green Supermix, 0.2 µl forward primer (10 pmol/µl), 0.2 µl reverse primer (10 pmol/µl), 3.6 µl double-distilled H₂O, 1 µl DNA template] in a StepOnePlus system (applied biosystems). The following primers were used: IL-17 forward: 5′-TACCAGCTGATCAGGACGAG-3′; IL-17 reverse: 5′-CATCAGGCACATGGATGGAA-3′; IFN-γ forward: 5′-ATTCATGAGCATCGCCAAGTTC-3′; IFN-γ reverse: 5′-TGACAGCTGGTGAATCACTCTGAT-3′; GAPDH forward: 5′-CCGAGGGCCCACTAAAGG-3′; GAPDH reverse: 5′-ATGGGAGTTGCTGTTGAAGTCA-3′. For qPCR, an initial denaturation step (95 °C, 30 s) was followed by 40 cycles of denaturation (95 °C, 15 s) and annealing/extension (60 °C, 1 min). The absence of unspecific amplification was determined by melt curve analysis. All reactions were run in triplicates.