5d starved_EdU index, pRPS6 Index, Cell cycle distribution pRPS6 +&- cells (Fig. 4AB & Fig. S5AC & Fig. S6C)

Juveniles were dissociated in pools of 15 polyps. Prior dissociation were labeled for 30 min with EdU (100uM, 2%DMSO) over several time points after feeding, and then dissociated 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, mOr2 was detected with anti-dsRed (Mouse, Takara Bio Clontech 632543) and goat-anti-mouse Alexa568, pRPS6 was detected with anti-pRPS6 (Ser235/236) (Rabbit, Cell Signaling Technology, 4858) and goat-anti-rabbit Alexa488.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，得到幼体细胞。在解离前，于喂食后的多个时间点用5-乙炔基-2'-脱氧尿苷（EdU，100μM，2%二甲基亚砜）标记30分钟，随后使用胰蛋白酶/甲醛进行解离与固定。细胞经1μg/ml的FxCycle Violet DNA染料（FxCycle violet DNA dye，Invitrogen）染色；采用Click-it反应（Click-it reaction）以Alexa647标记5-乙炔基-2'-脱氧尿苷（EdU）；使用抗dsRed抗体（anti-dsRed，小鼠源，Takara Bio Clontech 货号632543）与山羊抗小鼠Alexa568二抗检测mOr2蛋白；使用抗磷酸化核糖体蛋白S6（pRPS6，Ser235/236）抗体（anti-pRPS6 (Ser235/236)，兔源，Cell Signaling Technology 货号4858）与山羊抗兔Alexa488二抗检测pRPS6蛋白。采用BD LSRFortessa流式细胞仪（BD LSRFortessa，BD Life Sciences）完成流式细胞术（Flow cytometry）检测；借助FlowJoV10.8软件（FlowJoV10.8，BD Life Sciences），以细胞周期DNA染色结果为参照，通过与阴性对照对比Alexa568与Alexa647的荧光信号，对样本中的mOr2和5-乙炔基-2'-脱氧尿苷（EdU）阳性细胞进行鉴定。.wsp文件包含了FlowJo分析所用的样本信息、设门策略及细胞群数据；.fcs文件则为该.wsp文件中分析的单一样本流式细胞仪输出文件。