Mastermix kit

Total RNA from frozen tissues was extracted using the Total RNA Extraction Kit (SLNco, Cinoasia, China). Che concentration and quality of RNA samples were evaluated with a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Reverse transcription was carried out to obtain cDNA using the Master Mix kit (Takara, Shiga, Japan) following standard protocols. The mRNA levels of TNF-α, IL-6, and IL-1β in CAC colon samples and cells were assessed using a Step One Plus real-time PCR system (BIO-RAD, CFX96TM Real-time System, C1000TM Thermal Cycle, USA). Detailed information of the primer sequences employed in this study is summarized in Table 4.

Total RNA was extracted using TRIzol™ Reagent (Invitrogen) following the manufacturer’s instructions. Reverse transcription was performed using a MasterMix kit (Takara) following the manufacturer’s instructions. Quantitative polymerase chain reaction (qRT-PCR) was performed using a Universal SYBR Green MasterMix (Takara) on a QuantStudio 7 Flex real-time PCR system (Applied Biosystems). Gene expression was normalized to human GAPDH or zebrafish rpl13a. Primers were shown in supplementary table 1. For RNA-seq, whole transcriptome expression was measured using the NGS platform from Novogene according to Novogene’s suggested procedures for library construction and data analysis. Bioinformatic analysis was provided by Novogene (Beijing, China).

Total RNA was extracted with TRIzol reagent (Invitrogen, USA) according to a standard protocol. The concentration and quality of all the RNA samples were evaluated using a NanoDrop 2000 spectrophotometer (Thermo, USA), and the 260/280 and 260/230 values for all of the samples were above 1.8 and 1.9, respectively. Reverse transcription was performed with the MasterMix kit (Takara, Japan) and microRNA reverse transcription was performed with the TaqMan reverse transcription kit (Life Technologies, USA) following standard protocols. Quantitative PCR was performed using Universal SYBR Green Master mix (Applied Biosystems, USA), and microRNA qPCR was performed using TaqMan specific microRNA probes (Life Technologies) on a StepOnePlus real-time PCR system (Applied Biosystems). There were eight biological replicates per group. Additionally, eight technical replicates were used for each qPCR experiment. Gene expression was normalized to GAPDH, and microRNA expression was normalized to U6 unless otherwise stated. The primer list is included in the Supplementary File.