Sybr system

Patients' mucosal, DSS-induced colon tissue and CACO-2 cell were applied for total RNA extraction. Total RNA was reverse transcribed into cDNA by TOYOBO ReverTra Ace kit (TOYOBO). Subsequently, 0.5 ug cDNA was subjected by using the SYBR system (Takara) following the manufacturer's instructions in an CFX (Bio-Rad) real-time PCR machine. The mRNA expression level was calculated by the 2^−ΔΔCt method. The final values were normalized to the mRNA expression of β-ACTIN. The primers of β-ACTIN, ABIN1, RIPK1, MLKL, IL1B, IL8, TNF-α, and RIPK3 used for qRT-PCR are shown in Table 1.

Using a RNeasy Mini Kit (Qiagen, Valencia, California), total cellular RNA was extracted from cells. The eukaryotic and prokaryotic mRNA was enriched by Oligo (dT) beads or Ribo‐Zero^TM Magnetic Kit (Epicentre, Madison, WI, USA). Then, mRNA was transcripted into cDNA to be synthesized by DNA polymerase I, RNase H, dNTP, and buffer. RNA was purified and sequenced using Illumina Novaseq 6000 by Gene Denovo Biotechnology Co. (Guangzhou, China). Quantitative RT‐PCR was performed according to previous published papers using the SYBR system (Takara, China) [15]. The mRNA levels in cell and tissue lysates were normalized against 18S. The miRNA expression in cell and tissue lysates was normalized against U6. Sequences of primers used for qRT‐PCR in this study are listed in Table S2.

Treated mice colon tissue and cultured cells were freshly applied for total RNA extraction, using TRIzol reagent (Invitrogen) protocol. And then, quantitative real-time PCR was executed as described before. Briefly, 1 μg of the total RNA was reverse transcribed into cDNA and then, the cDNA was conducted by using the SYBR system (Takara) following the manufacturer's instructions in an ABI 7500 real-time PCR machine. The mRNA expression level was calculated by the 2 − ΔΔCt method; the final values were normalized to the mRNA expression of GAPDH. The primers of gapdh, FXRα, Shp, and lbabp used for qRT-PCR are shown in Table 1. In performing Western blot, proteins were separated on different concentrations of SDS-PAGE gel (7.5%-10%), and then, the separated protein was transferred from gel to nitrocellulose (NC) membranes and blotted with respective primary antibodies, followed by appropriate secondary antibodies and then detected with ECL chemiluminescent system The primary antibodies used in this study were anti-β-actin (13E5, CST), p-Smad2 (D43B4, CST), and anti-TGFBRI (ab31013 Abcam).