Enzyme Disaggregation for ChIP Experiments

Cell suspensions for ChIP experiments were obtained from frozen NT and TT of five CRC patients by enzyme disaggregation (ED), as previously described [23 (link)]. Briefly, thawed tissues were first washed with phosphate-buffered saline (PBS) and mechanically cut into small fragments (2–4 mm) using a surgical scalpel. Tissues were then suspended into RPMI-1640 with 1% penicillin/streptomycin and enzyme cocktail consisting of 1 mg/ml collagenase and 100 μg/ml hyluronidase type V (all from Sigma-Aldrich, UK) and incubated at 37 °C under slow rotation for 60 min. The resulting cell suspension was then passed through a 100 μm BD Falcon cell strainer (BD Biosciences, Oxford, UK), washed with serum free RPMI-1640, and resuspended in RPMI-1640 enriched with 10% FCS and 1% penicillin/streptomycin for further analyses.