title: ChIPmentation 100 cells
design: 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.