Beyochip enzymatic chip assay kit

A ChIP assay was performed according to the instructions of the BeyoChIP Enzymatic ChIP Assay Kit (#P2083S; Beyotime, Shanghai, China). Briefly, isolated primary LG epithelial cells were finely homogenized in 1% formaldehyde and then crosslinked at 37°C. The process of cross-linking was terminated by adding glycine solution at room temperature. Then, the cells were washed and collected with PMSF (1 µM). After the lysed cells were enzymatic, the fragments of chromatin (some for input) were incubated with Protein A/G Magnetic Beads/Salmon Sperm DNA and separated on a magnetic frame. Next, 2 µg of antibody against JunB (C-11) X (SC-8051; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added to the supernatant and incubated at 4°C overnight. The Protein A/G Magnetic Beads/Salmon Sperm DNA was added to the mixture. Then, the pellets were treated with elution buffer, and the supernatant was obtained after magnetic separation. For 2 hours, 5M NaCl was used for decrosslinking at 65°C. DNA was purified by phenol and chloroform extraction. The purifying DNA fragments were amplificated by nested PCR, and then the PCR products were subjected to electrophoresis in 1.5% agarose gel. The primers for ChIP are shown in the Table.

ChIP was performed using BeyoChIP™ Enzymatic ChIP Assay Kit (P2083S, Beyotime, China) according to the manufacturer's instructions. Anti-P65 antibody (10745-1-AP, Proteintech, USA) was applied to immunoprecipitate protein from AsPC-1 and MiaPaCa-2 cell lysates with A/G magnetic beads conjugated to the anti-P65 antibody. qPCR was performed to analyze the enrichment of MMP1. Primers used for ChIP-qPCR are listed in Supplementary Table S1.