GSM8119595: Emue_ATAC; Ephydatia muelleri; ATAC-seq
Two M. leidyi adult specimens were dissociated using CMFSW with 0.25% α-Chymotrypsin (Sigma-Aldrich, C8946). To isolate nuclei, dissociated cells were transferred into cold hypotonic ATAC lysis buffer adjusted for marine animals (10 mM Tris-HCl (pH 7.5), 35 mM NaCl, 3 mM MgCl2, 0,1% Tween-20, 0.01% NP-40, 0.01% digitonin, 70 µM Pitstop (Abcam, AB1206875MG)). Cell lysis was stopped after 2 min by adding marine ATAC wash buffer (10 mM Tris-HCl (pH 7.5), 35 mM NaCl, 3 mM MgCl2, 0,1% Tween-20, 1% BSA).  To isolate choanocytes of E. muelleri, 7 days post hatching sponges were fed with 0.5 µm fluorescent carboxylate-modified FluoSpheres (Invitrogen, F8813). After 10 min incubation, sponges were washed twice with Strekal's media, collected and dissociated for 15 min at 28 ºC using Protease XIV (Sigma-Aldrich, P5147). Cells were pelleted at 800 g for 5 min at RT and resuspended in Strekal's media with 2 mM EDTA (pH 8.0). Further dissociation and trituration of sponge tissue continued for additional 15 min at RT. Cells were filtered through 40 µm cell strainer, stained with 2 µg/ml Hoechst 33342, and FACS sorted. ATAC-seq libraries were prepared using Omni-ATAC protocol as described in Corces et al. 2017 (PMID: 28846090).