Hyluronidase type 5

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.

Enzyme disaggregation (ED) of frozen tumor and normal tissues from two breast cancer patients was performed on a roller mixer at 37 °C for 60 min. Briefly, after thawing the vials, tissues were first washed with phosphate buffer 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, 100 μg/ml hyluronidase type V, and 30 IU/ml of deoxyribonuclease I (all from Sigma-Aldrich, UK). Cell suspension was then passed through a 100-μm BD Falcon cell strainer (BD Biosciences, Oxford, UK) to remove debris and aggregates. Cells were then washed with serum-free RPMI-1640 and resuspended in RPMI-1640 enriched with 10% FCS and 1% penicillin/streptomycin and used for chromatin immunoprecipitation (ChIP) experiments.