*2.3. Real-Time-PCR*

Total *T. cruzi* RNA was extracted using a Direct-zol RNA Miniprep Kit (Zymo Research, Orange, CA, USA) from epimastigotes kept at RM, IDM, or IDM + Fe for 5 days (as indicated in the figure legends). The high-capacity cDNA reverse transcription kit was used to reverse-transcribe whole RNA (Thermo Fisher Scientific, Waltham, MA, USA). For RT-PCR, 100 ng/μL cDNA per well (15 μL total volume) was utilized, coupled with a 5 μM

primer mix and a 7 μL PowerUp SYBR green master solution (Thermo Fisher Scientific). Primers were designed using the Primer3 software, with predicted amplicon sizes of 100 pb each [37]. The primers for amplification are shown in Table 1. Gene expression data were normalized to an endogenous reference, β-tubulin. The expression ratios were determined using the threshold cycle (ΔΔCT) [38].

**Table 1.** Primer sequences for nine genes analyzed.

