cEdU Index Ad libitum (Fig. 2B & Fig. S2B)

Juveniles were dissociated in pools of 15 polyps. Polyps were continuously labeled with EdU (100uM, 2%DMSO) before dissociation, and fixed using Trypsin/formaldehyde. Cells were stained with 1μg/ml FxCycle violet DNA dye (Invitrogen), EdU was labelled with Alexa647 using a Click-it reaction, and mOr2 was detected with anti-dsRed (Rabbit, Takara Bio Clontech 632496) and goat-anti-rabbit Alexa568.
Flow cytometry was performed a BD LSRFortessa (BD Life Sciences) and the mOr2 and EdU positive cells in samples were determined based on Alexa568 and Alexa647 fluorescence in comparison with the negative controls on cells within the cell cycle (based on DNA staining) using FlowJoV10.8 (BD Life Sciences).
.wsp files contain the samples, gating strategy and cell populations used for the analysis in FlowJo
.fcs files represent the individual flow cytometry output files that were analysed in the .wsp file

以15个水螅体为一组进行解离，获取实验所需幼体。在解离前，持续使用含2% DMSO的100μM 5-乙炔基-2'-脱氧尿苷（EdU）对水螅体进行标记，随后采用胰蛋白酶-甲醛混合液完成固定。以1μg/mL的FxCycle Violet DNA染料（Invitrogen）对所得细胞进行染色；采用Click-it反应体系，通过Alexa647标记EdU；使用抗dsRed兔源抗体（Takara Bio Clontech，货号632496）以及山羊抗兔Alexa568二抗完成mOr2蛋白的免疫荧光检测。
采用BD LSRFortessa流式细胞仪（BD Life Sciences）开展流式细胞术检测；依托BD Life Sciences旗下的FlowJoV10.8软件，以基于DNA染色的细胞周期细胞群体为参照，通过对比阴性对照的Alexa568与Alexa647荧光信号，完成样本中mOr2阳性及EdU阳性细胞的鉴定与定量分析。
.wsp文件包含FlowJo分析所用的样本信息、门控策略及目标细胞群设门方案。
.fcs文件为单个流式细胞仪的原始输出文件，均通过对应的.wsp文件完成后续数据分析。