Single-cell analysis of allelic gene expression in pluripotency, differentiation and X-chromosome inactivation

We sequenced the mRNAs of embryonic stem cells (ESCs) cultured in different conditions. The two lines M (male) and F (female) used in this study were derived from E4 blastocysts of the same cross between a C57BL/6J (Mus musculus domesticus) and CAST/EiJ (Mus castaneus) male. mESCs were cultured in 2i and LIF as the ground state condition or in serum and LIF as the conventional condition. Epistem cell lines were also generated from the two lines by culturing them with Activin A and FGF2. In order to study more advanced development, we differentiated the two mESC lines through embryonic body formation to postmitotic motor neurons using retinoic acid and the smoothened agonist SAG. This differentiation process also results in the derivation of several types of interneurons. We picked single cells from all different conditions and generated sequencing libraries using the Smart-seq2 and Tn5 protocol. For simplicity, we designate the different condition as ES2i, ES, Epi and Neuron from hereon. We also obtained preimplantation inner cell mass and epiblast cells from E3.5 ICM (inner cell mass) and E4.5 blastocysts of the crossbred mice (male CAST/EiJ x female C57BL/6J) as well as postimplantation epiblast cells from E5.5 embryos of C57BL/6J mice Examination of gene expression profile in individual male and female embryonic stem cell lines along developmental progression本研究对不同培养条件下培养的胚胎干细胞（embryonic stem cells, ESCs）的信使RNA（messenger RNA, mRNA）进行了测序。本研究使用的M（雄性）与F（雌性）两株细胞系，均源自C57BL/6J（小家鼠Mus musculus domesticus亚种）与CAST/EiJ（Mus castaneus亚种）雄性个体交配获得的E4期囊胚。小鼠胚胎干细胞（mouse embryonic stem cells, mESCs）的培养分为两种体系：以2i与白血病抑制因子（leukemia inhibitory factor, LIF）组合作为原始态培养条件，或以血清与LIF组合作为常规培养条件。本研究还通过激活素A（Activin A）与成纤维细胞生长因子2（fibroblast growth factor 2, FGF2）培养上述两株细胞系，成功构建了Epistem细胞系。为探究更高级的发育进程，本研究借助视黄酸与smoothened激动剂SAG，通过拟胚体形成阶段，将两株mESCs诱导分化为终有丝分裂后的运动神经元；该分化过程同时可生成多种类型的中间神经元。本研究从所有培养条件中分选得到单个细胞，并采用Smart-seq2与Tn5转座酶建库方案构建测序文库。为简化表述，后续我们将不同培养条件分别命名为ES2i、ES、Epi与Neuron。本研究还从杂交小鼠（雄性CAST/EiJ × 雌性C57BL/6J）的E3.5囊胚内细胞团（inner cell mass, ICM）与E4.5囊胚中获取了植入前内细胞团与上胚层细胞，并从C57BL/6J小鼠的E5.5胚胎中获取了植入后上胚层细胞。本研究对不同性别胚胎干细胞系在整个发育进程中的基因表达谱进行了检测分析。