Trizol reagent

Total RNA was isolated using Trizol reagent (Magen, Guangzhou, China) and reverse transcribed using First Strand cDNA Synthesis Kit ReverTra Ace-α (TOYOBO, Osaka, Japan) according to the manufacturer’s recommendations. A 10 μL PCR-amplified reaction was performed according to the manufacturer’s instructions of LightCycler^® 480 SYBR Green I Master (Roche, Basel, Switzerland). All experiments were performed in triplicate. The sequences of the primers used in this study were as follows: MET forward, 5′- GGAGCCAAAGT-CCTTTCATCTGTAA-3′; MET reverse, 5′- GCAATGGATGATCTGGGAAATAAGAAG-AAT-3′; VEGFR2 forward, 5′- GGACTCTCTCTGCCTACCTCAC-3′; VEGFR2 reverse, 5′-GGCTCTTTCGCTTACTGTTCTG-3′; and GAPDH forward, 5′-GGAGCGAGATCCCTC-CAAAAT-3′; GAPDH reverse, 5′-GGCTGTTGTCATACTTCTCATGG-3′. The calculation of relative change in mRNA was performed using melting curve analysis and normalized to the housekeeping gene GAPDH.

The jejunal and ileal mucosa samples were ground into powder with liquid nitrogen, and total RNA was isolated using TRIZOL reagent (Magen, Guangzhou, China). The extracted RNA was reverse-transcribed into cDNA using a PrimeScript RT reagent kit with gDNA Eraser (TaKaRa Biotechnology Co., Ltd., Dalian, China). Real-time reverse transcription polymerase chain reaction (RT-PCR) assays were conducted using the SYBR^® Premix Ex Taq™ Kit (TaKaRa Biotechnology Co., Ltd.) on a LightCycler^® 480 II Real-Time PCR System (Roche, Basel, Switzerland) (16 (link)). The RT-PCR conditions were as follows: initial denaturation at 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 5 s, annealing at 60°C for 30 s, and a final extension at 72°C for 30 s. Primers were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd., (Shanghai, China) (Supplementary Table S4). Target gene expression was normalized against the housekeeping gene β-actin and calculated using the 2^-ΔΔCt method (17 (link)).

Total RNA was extracted from Jurkat E6.1 and SENP1^−/− T cells using TRIzol reagent (Magen) according to the manufacturer’s protocol. RNA was eluted with RNase-free water and quantified using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific). First-strand cDNA was synthesized by reverse transcription with Hifair III 1st Strand cDNA Synthesis Kit (gDNA digester plus, YEASEN, CHINA). Real-time PCR was run on a LightCycler 480 system (Roche) with Hieff qPCR SYBR Green Master Mix (YEASEN, CHINA) in triplicate. Real-time PCR primers sequences are shown in Supplementary Table S1. The relative quantity of mRNA was normalized with GADPH, using the comparative 2^–ΔΔCt method.

Total RNA of the colonic mucosa was extracted using the TRIZOL reagent (Magen, Guangzhou, China) according to the manufacturers' protocol. The total RNA (1,000 ng) was reversely transcribed into cDNA using a PrimeScrip RT reagent kit with gDNA Eraser (TaKaRa Biotechnology, Dalian, China). The two-step qRT-PCR analysis was performed on the LightCycler® 480 II Real-Time PCR System (Roche, Basel, Swiss) with the SYBR® Premix Ex Taq™ (TaKaRa Biotechnology, Dalian, China). Pig-specific primers were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd., China (Supplementary Table 2). The RT-qPCR was performed in a 10 μL reaction system, including 0.25 μL of each primer, 5.0 μL SYBR® Premix Ex Taq, 2.0 μL cDNA, and 2.5 μL of double-distilled water. The PCR cycling conditions referred to the instructions of SYBR Green Premix. The relative changes in gene expression were calculated by the 2^−ΔΔt method normalized to housekeeping gene β-actin.

The total RNA was extracted from the liver tissue using TRIzol^® Reagent (Magen R4801-02) according the manufacturer’s instructions (Magen, Guangzhou, China). RNA samples were detected based on the A260/A280 absorbance ratio with a Nanodrop ND-2000 system (Thermo Fisher Scientific Inc., Waltham, MA, USA), and the RIN of RNA was determined using an Agilent Bioanalyzer 4150 system (Agilent Technologies Inc., Santa Clara, CA, USA). Only qualified samples were used for library construction.

The cells were collected, and then the total RNA in the cells was extracted using TRIzol reagent (Magen, Guangzhou, China). Total RNA was subjected to reverse transcription (RT) using Transcription First Strand cDNA Synthesis Kit (Vazyme Biotech, Nanjing, China). Real-time quantitative PCR was then carried out in a CFX connect real-time system (Bio-Rad, Hercules, CA, USA) according to the operating instructions of SYBR Green Master Mix (Vazyme Biotech). According to the transcription level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, the gene to be detected was standardized and calibrated. Primer sequences for TRIM25 were used: forward, 5′-TCTGTAGGAGTCAAGGCTAAGGTG-3′, reverse, 5′-GTTGTGGGCGGTATTGTAGTCG-3′. The primer sequences for gRNA, GAPDH, IFNα, and IFNβ have been introduced in previous studies [64 (link)].

Total RNA was extracted from the tissues using TRIzol® Reagent according the manufacturer’s instructions (Magen). Paired-end libraries were prepared using a ABclonal mRNA-seq Lib Prep Kit (ABclonal, China) following the manufacturer’s instructions. The mRNA was purified from 1 μg total RNA using oligo (dT) magnetic beads followed by fragmentation carried out using divalent cations at elevated temperatures in ABclonal First Strand Synthesis Reaction Buffer. Subsequently, first-strand cDNAs were synthesized with random hexamer primers and Reverse Transcriptase (RNase H) using mRNA fragments as templates, followed by second-strand cDNA synthesis using DNA polymerase I, RNAseH, buffer, and dNTPs. The synthesized double stranded cDNA fragments were then adapter- ligated for preparation of the paired-end library. Adaptor-ligated cDNA were used for PCR amplification. PCR products were purified (AMPure XP system) and library quality was assessed on an Agilent Bioanalyzer 4150 system. Finally, the library preparations were sequenced on MGISEQ-T7 and 150 bp paired-end reads were generated. The data generated from BGI platform were used for bioinformatics analysis. All of the analyses were performed using an in-house pipeline from Shanghai Applied Protein Technology. The major software and parameters are as follows.