Trizol reagent

Quantitative real-time PCR Total RNA of carcinoid tissues was extracted with Trizol reagent (A2A0209, Accurate Biotechnology, China). cDNA was amplified with reverse transcription kit (A2A1386) was provided by Accurate Biotechnology Co. (China). The sequences of primers are in Supplementary Table 1. Gene expression levels were assayed by qRT-PCR using the Roche LightCycler^® 480 system (Roche, Basel, Switzerland) with the SYBR Green system (A2A1436, Accurate Biotechnology).

The total RNA was extracted from the PAMs (at least 1.0 × 10⁶ cells) with TRIzol™ Reagent (Accurate Biotechnology, Changsha, China) [28 (link)], and the cDNA was prepared with Super Script II Reverse Transcriptase (Thermo Fisher Scientific). Five pairs of primers for cloning the porcine DNMT1 gene (shown in Table S1) were designed on the basis of the porcine DNMT1 cDNA sequence reported in the GenBank database (NM_001032355.1). All the PCR products were sequenced with gene-specific primers. All the images of agarose gel electrophoresis were captured with the Image Lab Software version 5.1 (Bio-Rad Laboratories, Hercules, CA, USA).
The amino acid sequences of the human (NP_001124295.1), mouse (NP_001300940.1), porcine (NP_001027526.1), and cloned porcine DNMT1 proteins were aligned with Clustal V and edited with Genedoc. A phylogenetic tree was constructed from the available DNMT1 proteins with the neighbor-joining method in MEGA version 6.06 [29 (link)].

Total RNA was isolated from liver tissues using TRIzol reagent (Accurate Biology, Hunan, China). Subsequently, RNA samples were reverse transcribed into cDNA using the Evo M-MLV RT Kit (Accurate Biology, Hunan, China). The cDNA was used as the template for the real-time quantitative PCR (RT-qPCR) reaction. The primers were synthesized by Sangon (Shanghai, China). RT-qPCR was performed using SYBR^® Green Premix Pro Taq HS qPCR Kit (Accurate Biology, Hunan, China) according to the manufacturer’s instructions. RT-qPCR is carried out in the C1000Touch^™ Thermal cycle System (Bio-Rad, CA, United States). The relative expression for a particular gene was calculated using the method of 2^–ΔΔC. The primer sequences were listed in Table 1.

The RNA was extracted from liver, ileum, jejunum, and spleen using TRIzol Reagent (0.1 g tissue per 1 mL TRIzol, Accurate Biology, Changsha, China). The purity and concentration of extracted total RNA were determined using a NanoDrop 2,000 spectrophotometer (Thermo Fischer Scientific, Waltham, MA, USA). The total RNA (1 μg) was reverse-transcribed using the PrimeScript RT Reagent Kit with gDNA Eraser (Accurate Biology). Then 2.0 μL of cDNA template was mixed with the forward and reverse primers (0.25 μL, respectively), SYBR Green mix (5.0 μL), and RNase free water (2.5 μL). An RT-PCR analysis was performed on the Light Cycler^® 480 II Real-Time PCR System (Roche, Basel, Switzerland). The real-time PCR conditions were as follows: an initial step at 95°C for 5 min, 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. Pig-specific primer sequences are shown in Supplementary Table 2 (Sangon Biotech, Shanghai, China). Target gene expression was calculated using the 2^−ΔΔCt value (19 (link)), and β-actin was used as the internal control.

Total RNA was extracted from C57BL/6 mice and sEH KO mice by using TRIzol reagent (Accurate Biotechnology, Changsha, China), and the reverse transcription experiment was performed by using its corresponding kit to afford the cDNA. The PCR experiments were performed on an CFX96 Real-time System (Bio-Rad, California, USA) with MonAmp SYBR Green qPCR SuperMix (Monad, Shanghai, China). All measured values were normalized with β-actin according to the ΔΔCT method. These primers were listed in Table S1.

RNAs were extracted by TRIzol reagent (Accurate Biotechnology, Hunan, China), and reverse transcription of 1µg RNA was performed using PrimeScript RT Master Mix Kit (TaKaRa Bio. Inc., Dalian, China) to generate cDNA. Primers (Table S1) were synthesized by Tsingke Biotechnology (Beijing, China). The reaction system containing 2.0 μL diluted cDNA, 0.2 μM primers per pair, and 1×ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotechnology, Nanjing, China) was formulated, and was performed on the Real-Time PCR Detection System (BioRad, Hercules, CA, USA). After the detection, the specificity of primers was evaluated by the melting curve. β-actin was used as an internal control and the relative quantification of the target gene was assessed by arithmetic formulas (2^−ΔΔCt).

Total RNA from BUMPT cells and kidneys of C57BL/6J mice was extracted using a Trizol reagent (Accurate Biology, ChangSha, China) kit [8 (link), 26 (link), 27 (link)]. Total RNA (40 ng) was reverse-transcribed using Moloney Murine Leukemia Virus (M-MLV) Reverse Transcriptase (Accurate Biology). Real-time qPCR was used to examine the expression levels of miRNA, mRNA, and lncRNA by Bio-Rad (Hercules, CA, USA) iQ SYBR Green Supermix with Opticon (Accurate Biology, ChangSha, China) per the manufacturer’s instructions. The sequences of lncRNA ENSMUST_147219 were obtained from Ensembl database and those of miR-221-5p were obtained from miRDB. We used the following primers: ENSMUST_147219: 5′-TTTCACCCGCTTT GCCAAGTCTC-3′ (forward) and 5′-ATCCCTTCCCTGTCTCCTCAACTG-3′ (reverse); miR-221-5p: 5′-GCGACCTGGCATACAATGTAGAT-3′ (forward) and 5′-AGTGCAGGGTCC GAGGTATT-3′ (reverse); IRF6: 5′-ACAAACTGCTCTTCTATGGGCTTCTG-3′ (forward) and 5′-TCCTCCTCCTCATCTTCATCCACATC-3′ (reverse); β-actin: 5′-GGCTGTATTCCCCTCCATCG-3′ (forward) and 5′-CCAGTTGGTAACAATGCCATGT-3′ (reverse); and U6: 5′-CTCGCTTCGGCAGCACA-3′ (forward) and 5′-AACGCTTCACGAATTTGCGT-3′ (reverse). ΔCt values were used to carry out the relative quantification.