GSM8514501: HoiH23_ChIP_input; Cladtertia collaboinventa; ChIP-Seq
S. arctica and S. rosetta cells were double crosslinked with 1% formaldehyde and 3 mM DSG (Thermo Scientific, A35392). H.californensis specimens were flash-frozen in liquid nitrogen. Frozen tissues were then pulverized, cells and nuclei were collected by centrifugation for 15 min at 2,000 g. Collected cells were filtered through a 40 µm cell strainer and double crosslinked with 1% formaldehyde and 3 mM DSG. N. vectensis 1.5-2 month old specimens were crosslinked with 1 % formaldehyde under vacuum. Crosslinked tissues were dissociated into single cells in Ca2+/Mg2+-free artificial sea water in the presence of Protease XIV (Sigma-Aldrich, P5147). Dissociated cells were filtered through 40 µm cell strainer, stained with 2 µg/ml Hoechst 33342, and FACS sorted to isolate mOrange-positive neuronal cells. FACS-sorted cells were additionally crosslinked with 3 mM DSG. C. collaboinventa 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.