Ultrasybr mixture

Total RNA was isolated using Trizol reagent (Sigma Aldrich), RNA concentration was measured by a NanoDrop spectrophotometer (Thermo Fisher Scientific), and then cDNA was synthesized using HiFi Script cDNA Synthesis Kit (Cowin Biotech, Beijing, China) or miRcute Plus miRNA First-Strand cDNA Kit (Tiangen Biotech, Beijing, China).
The qRT-PCR was conducted by Ultra SYBR Mixture (Cowin Biotech). β-Actin (for circRNA) and U6 (for miRNA) were internal controls. Relevant primers involved in the current experiments were designed from Tsingke Biotechnology (Beijing, China), and the sequences of primers are shown in Table 2.

Total RNA was extracted from isolated PBMCs and DAKIKI cells with TRIzol Reagent (Invitrogen, CA, United States). Reverse Transcription Systems (for miRNA: CW2141, for mRNA: CW2569, CoWin Biosciences, Beijing, China) were used to reverse transcribe 1 μg of total RNA into cDNA. Levels of miR-98-5p, miR-152-3p, and C1GALT1, were then determined by qRT-PCR using the UltraSYBR Mixture (CW2601, CoWin Biosciences, Beijing, China) on a qRT-PCR detection system (Thermo Fisher, Pikoreal96, United States). Primers are shown in Supplementary Table S1. U6 snRNA and β-actin were used for normalization. Gene expression levels were calculated by the 2^−ΔΔCT method. All experiments were replicated three times.

RNA of peripheral blood mononuclear cells was extracted by EZ-10 Total RNA Mini-Preps Kit (Sangon Biotech, Shanghai, China) and subjected to reverse transcription using HiFiScript cDNA Synthesis Kit (CoWin Biosciences, Beijing, China). The messenger RNA levels were assessed by quantitative real‐time PCR using UltraSYBR Mixture (CoWin Biosciences, Beijing, China). β-actin was used as the internal control. The primers used in the study were as follows: β-actin, 5′‐AGA GGG AAA TCG TGC GTG AC‐3′ (sense) and 5′‐CAA TAG TGA TGA CCT GGC CGT‐3′ (antisense); CCR7, 5′‐TGT ACG AGT CGG TGT GCT TC ‐3′ (sense) and 5′‐GGT AGG TAT CCG TCA TGG TCT TG‐3′ (antisense); KLRK1, 5′‐ACT CAG AGA TGA GCA AAT GCC‐3′ (sense) and 5′‐CAG GTT GAC TGG TAG TTA GTG C‐3′ (antisense); TIGIT, 5′‐ CCA CAG CAG GCA CGA TAG ATA ‐3′ (sense) and 5′‐CCA CCA CGA TGA CTG CTG T‐3′ (antisense); Slamf1, 5′‐CAG AAA TCA GGG CCT CAA GAG‐3′ (sense) and 5′‐ CAT GCC ACC CCA GGT CAA C ‐3′(antisense).

Total RNA was isolated by using TRIZOL reagent (Invitrogen) and the phenol-chloroform extraction method according to manufacturer protocols. The cDNA was synthesized using a First-Strand cDNA Synthesis Kit (Invitrogen). Quantitative real-time PCR was performed on an ABI 7300 instrument with UltraSYBR Mixture (CoWin Biosciences) according to manufacturer instructions with the following primers: GAPDH, forward: 5′-AGGCTGAGAACGGGAAGC-3′, reverse: 5′-CCATGGTGGTGAAGACGC-3′; TRAP1, forward: 5′-CGCAGCATCTTCTACGTGC-3′; reverse: 5′-CTGATGAGTGCGCTCTCC-3′.

Total RNA was extracted from cells using Trizol reagent (Invitrogen™, Thermo Fisher Scientific) according to the manufacturer’s instructions. After quantification, cDNA was obtained using the Reverse Transcription System (Promega, Corporation, Fitchburg, MA, USA), with oligo (dT) primer for mRNA and stem-loop reverse transcription primer for miRNA. The mRNA expression level of genes was examined by RT-qPCR using UltraSYBR Mixture (CoWin Biosciences, Beijing, China) with the following pairs of primers: PPARγ forward, 5′-TGTGGGGATAAAGCATCAGGC-3′, and reverse 5′-CCGGCAGTTAAGATCACACCTAT-3′; C/EBPα forward, 5′-GCTGGAGTTGACCAGTGACA-3′, and reverse 5′-AAACCATCCTCTGGGTCTCC-3′; C/EBPβ forward, 5′-GCAAGAGCCGCGACAAG-3′, and reverse 5′-GGCTCGGGCAGCTGCTT-3′; FABP4 forward, 5′-ACACCGAGATTTCCTTCAAACTG-3′, and reverse 5′-CCATCTAGGGTTATGATGCTCTTCA-3′; c-Met forward, 5′-ACCAAGTGCTCCTGACATCC-3′, and reverse 5′-GTGAGGTGTGCTGTTCGAGA-3′;GAPDH forward, 5′-AAGAAGGTGGTGAAGCAG-3′, and reverse 5′-GAAGGTGGAAGAGTGGGAGT-3′; U6 snRNA forward, 5′-CTCGCTTCGGCAGCACA-3′, and reverse 5′-AACGCTTCACGAATTTGCGT-3′; mmu-miR-206-3p forward, 5′-ACACTCCAGCTGGGTGGAATGTAAGGAAGTGT-3′, and reverse 5′-TGGTGTCGTGGAGTCG-3′. Relative quantification (2^−ΔΔCt) method was used to analyze the data, GAPDH mRNA was used as reference for mRNA quantification, and U6 snRNA was used for miRNA.