Figure 1

Optimization of sonication conditions to yield ~2000-bp chromatin fragments and of formaldehyde concentrations for efficient crosslinking. A: 2 × 10⁷ cells of Chlamydomonas strain CF185 were sonicated 0, 5, 10 and 20 times for 10 seconds. DNA was extracted with phenol/chloroform/isoamylalcohol, separated on a 1.5% agarose gel and stained with Ethidium bromide. B: 2 × 10⁷ cells of strain CF185 were incubated for 10 min with formaldehyde (HCHO) at the concentrations indicated. After sonication of cells crosslinks were reversed by over-night incubation at 65°C. DNA was extracted and visualized as in (A). C: whole-cell proteins corresponding to 2 μg chlorophyll from strain cw15-325 were separated on a 15% SDS-polyacrylamide gel and analyzed by immunoblotting using antibodies against histone H3. D, E: ChIP was done with preimmune serum (mock) and with antibodies against histone H3 using chromatin as input that was crosslinked with formaldehyde at the indicated concentrations. Precipitated DNA was amplified using primers targeting the HSP70A promoter (D) or the RBCS2 promoter (E) and visualized as in (A). NTC-non-template control; TC-template control based on 10% input DNA.