Cdna synthesis kit

Total RNA was prepared with the Axygen^® AxyPrep Multisource RNA Miniprep Kit (Corning, USA) and cDNA was transcribed with the cDNA Synthesis Kits (Bio-Rad, USA) or the MicroRNA Reverse Transcription Kit (Takara, Japan). The SYBR^® Green Master Mix (Bio-rad, USA) or miRcute miRNA qPCR Detection Kit was used for the qRT-PCR (TIANGEN, China). For miR-1-3p, miR-4443, and miR-516a-5p, internal reference was U6. The primers were produced by Sangon Biotechnology Inc. (Shanghai, China). The sequence of primers was as follows:
U6 (Forward : CTCGCTTCGGCAGCACA, Reverse : AACGCTTCACGAATTTGCGT), hsa-miR-1-3p (Forward : CGGGCTGGAATGTAAAGAAG, Reverse : CAGCCACAAAAGAGCACAAT), hsa-miR-4443 (Forward : CGGGCTTGGAGGCGT, Reverse : CAGCCACAAAAGAGCACAAT), hsa-miR-516a-5p (Forward : CGGGCTTCTCGAGGAAAGAAG, Reverse : CAGCCACAAAAGAGCACAAT), hsa-miR-561-5p (Forward : CGGGCATCAAGGATCTTAAA, Reverse : CAGCCACAAAAGAGCACAAT).

Lipopolysaccharide (E. coli 055: B5), SDS, EDTA, hydroxylamine hydrochloride, thiobarbituric acid, RNA later, Bradford reagent, and protease inhibitor cocktail were procured from Sigma-Aldrich (St. Louis, MO, United States). TRIzol was purchased from Invitrogen, California, United States. de Man, Rogosa, and Sharpe (MRS) agar, glass slides, paraffin, hematoxylin, eosin, alcian blue, glycerol, hydrogen peroxide (H₂O₂), sodium bicarbonate (NaHCO₃), sodium carbonate (Na₂CO₃), sulfosalicylic acid, formalin, potassium chloride (KCl), and other chemicals were from HiMedia Laboratories, Mumbai, India; mouse-specific lipocalin-2, IL-6, IL-1β, and TNF-α ELISA kits were procured from Ray Biotech, Norcross, United States. SYBR-Green master mix and cDNA synthesis kits were purchased from Bio-Rad, United States. HPLC with a diode array detector was from Agilent Technologies, Singapore. NucleoSpin DNA Stool kits were procured from Macherey-Nagel, Düren, Germany. Antibodies (occludin, zona occludin, claudin, GAPDH, Muc-4, and Alexa Fluor 488) from Cell Signaling Technology (Danvers, MA, United States). HRP (horseradish peroxidase)-conjugated secondary antibody and DNase treatment AM1906 were purchased from Thermo Fisher, Leica CTR6, and Leica Biosystems, Germany. A confocal microscope was purchased from Carl Zeiss LSM-880, Germany.

Isolation of RNA, cDNA synthesis, and quantitative PCR were performed based on the manufacturers’ instructions for QIAGEN (RNeasy Kits), Thermo Fisher Scientific (cDNA synthesis Kits), and Bio‐Rad (SYBR Green Master Mix), respectively. Gene transcript levels were determined using the ΔΔC_t method for multiple‐reference gene correction. β‐Actin and GAPDH were used as references. qPCR primer sequences are provided in Table EV1.

RNA isolation, cDNA synthesis, and quantitative PCR were performed based on manufacturer’s instructions for QIAGEN (RNeasy Kits), Thermo Fisher Scientific (cDNA synthesis Kits), and Bio-Rad (SYBR Green Master Mix), respectively. Reactions were performed in duplicate and a relative amount of cDNA was normalized to house-keeping genes indicated on figure legends using the ΔΔCt method. Sequences of primers used for qPCR are listed in Supplementary Table S2.