Rox reference dye

Total RNA was extracted with TRIzol (Invitrogen, US) from each tissue sample followed by the instructions. 300 ng RNA of each sample was used to perform reverse transcription reaction using the reverse transcription kit FSQ-301 (TOYOBO, Shanghai, China) according to the instructions. The reverse transcription primers were 5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCCCCTATC-3′ (miR-155), oligo(dT) (CTLA-4 gene and internal reference β-actin) and 5′-AACGCTTCACGAATTTGCGT-3′ (internal reference U6), respectively. Quantitative real-time PCR (qRT-PCR) was used to analyze the expression levels of different targets. The qRT-PCR primers were as follows: miR-155: 5′-ACACTCCAGCTGGGTTAATGCTAATCGTGA-3′ and 5′-TGGTGTCGTGGAGTCG-3′; U6: 5′-CTCGCTTCGGCAGCACA-3′ and 5′-AACGCTTCACGAATTTGCGT-3′; CTLA-4: 5′-TGCCGAAGTAATGGAAGTGA-3′and 5′-TTTCAGTGAACTTGTCGCCT-3′; β-actin: 5′-TATGTGCAAGGCCGGTTTC-3′ and 5′-CACCAACGTAGCTGTCTTTCTG-3′. All qRT-PCR systems were 10 µL including: 5  µL  1 ×SYBR Green I (TOYOBO, Shanghai, China), 0.2  µL  50 ×ROX reference dye (TOYOBO, Shanghai), 0.3  µL of each primer, 1.2 µL cDNA and 3  µL ddH₂O. QRT-PCR processes were: 95 °C/1 min; 45 cycles of 95 °C/15s, 60 °C/30s, 72 °C/30s. The qRT-PCR system doses and reaction procedures of U6, β-actin and CTLA-4 were the same as miR-155.

Total RNA was extracted from different tissues using TRIzol (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Agarose gel electrophoresis (AGE) was used to estimate the integrity of total RNA, and a DU800 spectrophotometer (Beckman Coulter, Miami, FL, USA) was used to quantify the total RNA. Briefly, 300 ng of RNA of each sample was reverse-transcribed using the reverse transcription kit FSQ-301 (TOYOBO, Shanghai, China) following the manufacturer’s instructions. qRT-PCR amplification efficiency was assessed by varying the cDNA template concentration to obtain a relative quantitative standard curve, and specificity was assessed by melting curve. qRT-PCR was performed in three technical iterations with a 10 μL reaction mixture, which contained 5 μL of 2× SYBR Green I (TOYOBO, Shanghai, China), 0.2 μL of 50× ROX reference dye (TOYOBO, Shanghai, China), 0.3 μL of each primer, 1 μL of cDNA, and 3.2 μL of RNA-free water. U6 was chosen as an endogenous control for miRNAs. The qRT-PCR procedure was as follows: 95 °C for 1 min, followed by 40 cycles at 95 °C for 15 s, 60 °C for 30 s, 72 °C for 30 s, and s final extension for 30 s at 72 °C. All primer sequences are provided in Table 1.

Total RNA was extracted using the TRIzol reagent (Thermo Fisher Scientific, 15596018). RNA was reverse transcribed using a ReverTra Ace® qPCR RT Master Mix with gDNA Remover Kit (TOYOBO, FSQ‐301). QuantStudio 6 Pro Real‐Time PCR System (Thermo Fisher Scientific) was used to perform the real‐time quantitative PCR analysis with THUNDERBIRD SYBR^® qPCR Mix (TOYOBO, QPS‐201) plus 50 × ROX reference dye (TOYOBO, QPS‐201). All these kits were used in accordance with the manufacturer's guidelines. Equal loading was achieved by amplifying GAPDH mRNA. The primers used were listed in Table S3. All reactions were conducted in triplicate.

Total RNA was isolated from piglet stomach tissue using TRIzol Reagent (Thermo Fisher Scientific), according to the manufacturer’s instructions. The RNA was reverse transcribed into cDNA using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO, Osaka, Japan), according to the manufacturer’s instructions. The sequences of primers used for real-time PCR were as follows: AMCase forward primer 5ʹ-TGACTTCACAGGCACTTTCT-3ʹ, AMCase reverse primer 5ʹ-CGGTGCAACTTGTGCTATTC-3ʹ; Chit1 forward primer 5ʹ-GTCAACTCAGCCATCAGGTT-3ʹ, Chit1 reverse primer 5ʹ-CAAGGTCAAGGCCATCAAA-3ʹ; and GAPDH forward primer 5ʹ-ACCTCCACTACATGGTCTACA-3ʹ, GAPDH reverse primer 5ʹ-ATGACAAGCTTCCCGTTCTC-3ʹ^{24 (link)}. A StepOnePlus Real-Time PCR system (Applied Bioscience, Waltham, MA, USA) was used for real-time PCR, and each reaction mixture consisted of 0.4 µl of 50 × ROX reference dye (TOYOBO, Osaka, Japan), 10 µl of THUNDERBIRD SYBR qPCR Mix (TOYOBO, Osaka, Japan), 2 µl of DNA template, 0.5 µM of forward primer, 0.5 µM of reverse primer, and 5.6 µl of sterile water, with a total volume of 20 µl. Each reaction was performed in triplicate. The PCR cycling conditions were as follows: initial denaturation at 95 °C for 1 min; 40 cycles of denaturation at 95 °C for 15 s, and extension at 60 °C for 1 min.

Target tissues were homogenised and RNA extraction was performed with TRIzol (Invitrogen, Waltham, MA, USA)/chloroform method. Nanodrop analysis was performed to determine the concentration of RNA. RNA was diluted with RNAse-free purified water to a concentration of 1000 ng/μL. cDNA was generated from the extracted RNA (1000 ng/μL) using ReverTra Ace^® qPCR RT Master Mix (Toyobo Co. Ltd., Osaka, Japan) following the manufacturer’s instructions. 1 μL of RNA was used with 19 μL of qPCR RT Master Mix. cDNA was reverse transcribed by using a thermal cycler on a cycle running at 37 °C for 15 min, 98 °C for 5 min, and finally 4 °C for 5 min. The qPCR mixture included THUNDERBIRD^® SYBR^® qPCR Mix, Rox reference dye (Toyobo Co. Ltd., Osaka, Japan), and primers. The qPCR was performed according to the manufacturer’s instructions. Expression levels were all relative to those of β-actin used as an endogenous control. Experiments were designed and analysed by using Stepone Software v.2.2.2. The qPCR was performed as follows: 95 °C for 60 s, 95 °C for 3 s, 60 °C for 30 s, repeated for 40 cycles, and finally a melting curve was obtained.