title: GSM8119608: Tadh_ChIP_H3K4me3; Trichoplax adhaerens; ChIP-Seq
library_construction_protocol: Capsaspora owczarzaki cells were double crosslinked with 1% formaldehyde and 3 mM DSG (Thermo Scientific, A35392).  Mnemiopsis leidyi specimens were dissociated into single cells in Ca2+/Mg2+-free artificial sea water by triturating every 2 min for a total of 10 min. Dissociated cells were filtered through a 40 µm cell strainer and double crosslinked with 1% formaldehyde and 3 mM DSG. Seven days post hatching sponges Ephydatia muelleri were fed with 0.5 µm fluorescent carboxylate-modified FluoSpheres (Invitrogen, F8813). Next, animal tissues were dissociated into cells and double crosslinked with 1% formaldehyde and 3 mM DSG. Crosslinked cells were filtered through 40 µm cell strainer, stained with 2 µg/ml Hoechst 33342, and FACS sorted to isolate choanocytes. Trichoplax adhaerens specimens were dissociated into single cells in Ca2+/Mg2+-free artificial sea water. Dissociated cells were filtered through a 40 µm cell strainer and double crosslinked with 1% formaldehyde and 3 mM DSG. Chromatin immunoprecipitation was performed as previously described by Lara-Astiaso et al. (PMID: 25103404) with following modifications. For each species, 100 ng chromatin was used for immunoprecipitation. The pool of chromatin was incubated for 14 - 16 hours at 4 ºC with 6 µL (3.4 µg) anti-H3K4me2 (Abcam, ab32356), 2.5 µL anti-H3K4me3 (Millipore, 07-473), 5 µL (5 µg) anti-SMC1 (ThermoFisher , A300-055A) or 2 µL (2 µg) anti-H3 (Abcam, ab1791).