Forward and reverse primer

Quantitative real-time PCR was performed on CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, United States). Amplification mixture consisted of PowerUpTM SYBR^® Green master mix (Thermo Fisher Scientific), 10 μM forward and reverse primers (Invitrogen, Carlsbad, CA, United States) and approximately 1.5 μl of cDNA template. Primer sequences were obtained from the literature and checked for their specificity through in silico PCR. The forward and reverse primers are shown in Table 1. Amplification was carried out with an initial denaturation step at 95°C for 2 min followed by 45 cycles of 95°C for 10 s, 55°C for 30 s and 60°C for 30 s, then 65°C for 2 min in 10 μl reaction volume. All reactions were run in duplicate and the results were averaged from 6 independent studies. qPCR was quantified in two steps. First, β-actin levels were used to normalize target gene levels (ΔCycle threshold (ΔCt) = Ct_{target gene} − Ct_β–actin, target gene level = 2−ΔCt). Second, the target gene levels of sevoflurane group were presented as the percentage of those of control group, and 100% of the target gene levels referred to the control levels.

The 357 bp fragment which harbors GG and TT genotypes from ca. $-$ 677 to ca. $-$ 320
was amplified with primer H. The 20  $\mathrm{\mu }$ L amplification system
consisted of 200 ng DNA templates, 10  $\mathrm{\mu }$ L rTaq Premix (Takara, Dalian,
China), 1  $\mathrm{\mu }$ L of forward and reverse primers (10  $\mathrm{\mu }$ mol) (Invitrogen,
Shanghai, China), and 7  $\mathrm{\mu }$ L ddH ${}_{\mathrm{2}}$ O. The cycling protocol was 5 min at
94  ${}^{\circ }$ C, 35 cycles of 94  ${}^{\circ }$ C for 30 s, 64  ${}^{\circ }$ C
annealing for 30 s, and 72  ${}^{\circ }$ C for 30 s, with a final extension at
72  ${}^{\circ }$ C for 7 min. The forward primer was added NheI restriction
enzyme cutting site, and reverse primer was added HindIII restriction
enzyme cutting site. The two genotype fragments were then cloned into the
pGL3-basic vector (Promega, USA) separately. Twenty-four hours before
transfection, 293T cells were seeded in each well of 12-well plates.
The 293T cells were co-transfected with the constructed reporter plasmid and pRL-TK plasmid (Promega, USA). The transfection system consisted of 1  $\mathrm{\mu }$ g TT
or GG genotype reporter plasmid, 0.05  $\mathrm{\mu }$ g pRL-TK plasmid, 100  $\mathrm{\mu }$ L
OPTI-MEM, and 3  $\mathrm{\mu }$ L Lipofectamine^® 2000 Reagent. Experiments were performed in biological triplicate.
Twenty-four hours after transfection, cells were lysed in passive lysis
buffer (Promega, USA). Firefly luciferase activity and Renilla luciferase
activity were measured according to the manufacturer's protocol in three
independent experiments (Promega, USA).

Extracted DNA (5 µl) was added to a master mix (20 µl) to give a total reaction volume of 25 µl per sample. The master mix was prepared by mixing 12.5 µl of Bio-Rad iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, California), 5.5 µl of DEPC-treated nuclease free sterile water (Fisher Scientific, Pittsburgh, Pennsylvania), and 1.0 µl (6.2 mM) each of both forward and reverse primers (Invitrogen, Carlsbad, California). The primers target the 18 s rRNA gene of the parasite as shown in Table 1 (GenBank no. AF164102). The reaction was performed in a Bio-Rad iCycler iQ multicolor PCR Detection System using iCycler software (version 3.0). Amplification consisted of 15 min at 95°C followed by 40 cycles of 15 sec at 95°C, 15 sec at 52°C, and 20 sec at 72°C, followed by 0.5-degree increments for 10 sec starting at 75°C and ending with 95°C for the Melt Curve. Fluorescence was measured during the annealing step of each cycle. Ct values of each run were compared to standards with known amounts of C. parvum DNA and log transformed into number of organisms per mg of stool sample.

The significances of genes changes were calculated by −log10 (p-value), and higher −log10 (p-value) indicated that the gene was with more significant changes. We selected the top differentially expressed genes for qPCR verification.
qPCR was performed on the CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Amplification mixture consisted of PowerUpTM SYBR^® Green master mix (Thermo Fisher Scientific), 10 μM forward and reverse primers (Invitrogen, Carlsbad, CA, USA) and approximately 1.5 μl of cDNA template. Primer sequences were obtained from the literature and checked for their specificity through in silico PCR. The forward and reverse primers are shown in Table 1. Amplification was carried out with an initial denaturation step at 95°C for 2 min followed by 45 cycles of 95°C for 10 s, 55°C for 30 s and 60°C for 30 s, then 65°C for 2 min in 10 μl reaction volume. All reactions were run in duplicate and the results were averaged from six independent studies. qPCR was quantified in two steps, first, β-actin levels were used to normalize target gene levels [ΔCycle threshold (ΔCt) = Cttarget gene—Ctβ-actin, target gene level = 2-ΔCt]. Second, the target gene levels of the sevoflurane group were presented as the percentage of those of the control group, and 100% of the target gene levels referred to the control levels.