C4 column

To generate Alexa Fluor 488 labeled histone H2B (H2B-Alexa488), histone H2B with T112C mutation was dissolved in reaction buffer (25 mM HEPES, pH 7.5, 6 M guanidine, 5 mM TCEP) to a final concentration of 0.5 mM. Then five molar equivalents of Alexa-488-C₅-maleimide (Invitrogen) was added to the reaction mixture. Keep the reaction for 4 h at 37°C. The fluorophore labeled histone was purified by HPLC using preparative C4 column (25 mm × 250 mm, 10 μm, Grace). The purity and identity of the product was confirmed by LC–MS. Deconvolution result was obtained by UniDec software (25 (link)) (Supplementary Figure S4). Same method was applied to generate Cy5 (Cy5 Maleimide Mono-reactive Dye, GE) labeled histone H2A at K119 position (Supplementary Figure S5).

In order to identify the antibiofilm components of ES products, SPE eluate was fractionated using a reverse phase (RP)-HPLC chromatography (Beckman System Gold, USA) coupled with a C18 column (250 mm × 4.6 mm; 5 μm) (Grace, USA) at a flow rate 0.3 mL/min, by using a gradient from 0 to 90% (v/v) acetonitrile (containing 0.1% (v/v) trifluoroacetic acid), during 70 min. The fractions were lyophilized and dissolved in 100 μL of TSB and tested for antibiofilm activity against S. aureus. The active fraction was then fractionated using a C4 column (250 mm × 4.6 mm; 5 μm) (Grace, USA) and tested under the same conditions. The purity of active fractions was checked by electrophoresis on 16.5% Tricine-SDS-PAGE gel using a Mini-Protean II electrophoresis cell (Bio-Rad, CA, USA). Protein bands were detected after staining with Coomassie Brilliant Blue R-250.

LC/MS/MS analyses were performed using an Agilent Triple Quadrupole LC/MS/MS (Santa Clara, CA). Mobile phases (flow rate: 0.8 mL/min) were A: H₂O + 0.1% formic acid (FA) and B: 50:50 ACN:MeOH + 0.1% FA, with a gradient sequence given in Table 3. A Vydac C4 column (Grace, Columbia, MD) was used as stationary phase. The charging voltage was 1000 V and the capillary voltage was 4000 V. The nebulizer gas temperature was 325 °C, the gas flow was 8 L/min, and the nebulizer pressure was 45 psi. The sheath gas heat and flow rate was 250 °C and 7 L/min, respectively. Injection volumes were 0.5 μL and the total run time was 15 min. Transitions monitored were m/z 492-386 for cholesterol and m/z 499-393 for d₇-cholesterol. Ion polarity was positive. Three sequential injections of wash solution (ACN or MeOH) were done after every six experimental samples. Liver samples were extracted, derivatized, and analyzed in two separate experiments and found to differ by ≤5.8%. All subsequent cholesterol analyses were done once.