*4.8. Chromatin Immunoprecipitation (ChIP)*

Chromatin Immunoprecipitation (ChIP) was performed using the Chromatrap ChIP-Seq kit (Cat No: 500189, Porvair, Ashland, VA, USA) using either JARID2 or EZH2 antibodies. Approximately 70% confluent Ishikawa cells were treated with 1% PF from patients for 48 h in a DMEM/F12 media containing 1% charcoal-stripped FBS. Proteins were cross-linked by adding formaldehyde (0.75% by volume) and allowing for a 10-min incubation at room temperature. Glycine (0.5M) was added and incubated for an additional 10 min. Cells were twice rinsed with PBS, collected in 1 mL PBS and pelleted by centrifugation. All other buffer and components used were obtained from the kit. Protocol v1.5 of the manufacturer's instructions was followed (Porvair, Ashland, VA, USA). Cells were suspended in 800 µL of hypotonic buffer before being centrifuged and the nuclear pellet was separated and resuspended in 400 µL of pre-warmed lysis buffer. Sonication was performed using a Covaris ME220 (SKU: 500506, Woburn, MA, USA). Each sample was aliquoted into 3 different tubes prior to sonication. Each tube was sonicated for 100 s and sheering efficiency was verified using an agarose gel as a smear of DNA fragments between 100–500 bp in length. ChIP-grade anti-JARID2 antibody (Cat No: 13594, Cell Signaling, Danvers, MA, USA) and EZH2 (Cat No: 5246S, Cell Signaling, Danvers, MA, USA) was used for antibody precipitation. DNA concentration was determined by NanoDrop 2000 spectrophotometer. The Human Polycomb & Trithorax Complexes EpiTect ChIP qPCR Array (Cat No: 334211 GH-506A, Qiagen, Gaithersburg, MA, USA) consisting of primers for genes belonging to the polycomb and trithorax complexes (core, alternate, and additional components), as well as polycomb co-factors such as PHD finger protein 19 (PHF19) and heterochromatin (CBX) proteins was run for all samples. Percent enrichment and further statistical analysis was calculated using algorithm provided by Qiagen/SA Biosciences (Gaithersburg, MA, USA).
