ChIPmentation 10e4 cells
Chromatin preparation was performed using the Diagenodes ChIPmentation Kit for Histones, Cat. No. C01011009 and protocol with minor modifications. Two times 10,000 miracidia (1,000,000 cells based on the observation that 1 miracidium is composed of 100-120 cells) were resuspended each in 500 L 1x HBSS and crashed with a plastic pestle in an Eppendorf tube on ice during ~1 min. For cross-linking, 13.5 L of formaldehyde were added and tubes were incubated for 10 min at room temperature with occasional inversion. To stop cross-linking fixation, 57 L of glycine were added and samples were incubated for 5 min at room temperature. Samples were centrifuged at 500xg, at 4C, for 5 min. The pellet was resuspended in 2x1 mL of ice-cold Lysis Buffer iL1, combined and homogenized in a Dounce (pestle A) on ice for 5 min. After another centrifugation (500g, 5 min, 4C), the supernatant was discarded and the pellet was resuspended in 1 mL of ice-cold Lysis Buffer IL2 and centrifuged (500xg, 5 min, 4C). The supernatant was discarded and the pellet was resuspended in 100 L of complete Shearing Buffer iS1 for each tube. Samples were sonicated with the Bioruptor Pico (Diagenode, Cat. No. B01080010) for 5 cycles (30s ON and 30s OFF). After transfer into new tubes, samples were centrifuged (16,000g, 10 min, 4C). The supernatants were transferred into a new single tube (200 l total) and 20 l iS1 were added yielding a total volume of 220 l. The procedure was done in duplicate (named L and R in the following). Serial dilutions were done in iS1 to produce 100 l equivalents of 10,000 miracidia (106 cells), 1,000 miracidia (105 cells), 100 miracidia (104 cells), 50 miracidia (5,000 cells), 10 miracidia (103 cells), 5 miracidia (500 cells) and 1 miracidium (100 cells) or 100 l iS1 as negative control.