Beyochip chip assay kit

ChIP assay was performed using BeyoCHIPTM CHIP Assay Kit (Beyotime) according to the manufacturer's instructions. T‐DCs were fixed via the addition of formaldehyde (final concentration of 1%) for 10 min, then administering glycine was to neutralize extra formaldehyde. Cells were washed with PBS, lysed with SDS Lysis Buffer for 10 min, then sonicated under 4°C to shear the cross‐linked DNA to 200–1000 bp. The cross‐linked protein‐DNA was immunoprecipitated with magnetic beads, and protein‐DNA complexes were precipitated using IgG and anti‐ZNF148 antibody (Abclonal) overnight. After that, the eluted DNA fragments were detected with PTX3 promoter‐specific primers and SYBR premix.

Total RNA was isolated from control or siYY1 LOVO cells, and were sent for RNA-sequencing, which was performed in a Genergy Biotechnology company (Shanghai, China). Chromatin immunoprecipitation (ChIP) assay was performed using the BeyoChIP^TM ChIP Assay Kit (Beyotime, China) in HCT116, LOVO, and DLD1 cells according to the manufacturer’s instructions. DNA was purified using a PCR purification kit (Tiangen, China) and prepared for PCR analysis. Then, agarose gel electrophoresis of the PCR product was carried out in 2% agarose gel containing DNA Gel dye. The primers used for ChIP-PCR analysis were listed in Supplementary Methods and Table S2.

ChIP assays were carried out according to the instructions of the BeyoChIP™ ChIP Assay Kit (Beyotime, Shanghai, China, P2080S). Briefly, the cells were subjected to fixation in 1.0% v/v formaldehyde for 15–20 min at room temperature, followed by quenching with 100 mM glycine. The cells were subsequently disrupted using a mini beadbeater, and the crosslinked chromatin was fragmented by sonication using a Bioruptor (7 cycles of 30 s on/off). The resulting extracts were then incubated for either 2 h or overnight at 4 ˚C with magnetic Prot A beads that were conjugated with a polyclonal antibody against DEC1 (Proteintech, Wuhan, China, 17895-1-AP). Sub-sequently, the reverse cross-linking process was conducted in Tris-EDTA buffer consisting of 100 mM Tris pH 8.0, 10 mM EDTA, and 1.0% v/v SDS at a temperature of 65 °C overnight. Following a proteinase K treatment of 2 h, the samples were subjected to a cleanup process, and the enrichment of DEC1 was determined through real-time PCR using SYBR green mix. The chromatin DNAs that were precipitated underwent Quantitative PCR using the identical procedure as that of qRT-PCR. The percentage of input DNA was utilized to determine the relative enrichment of the DNAs.