最近这篇Science文章不错，Multifaceted SOX2-chromatin interaction underpins pluripotency progression in early embryos - 15 December 2023
需要复刻里面的一些思路、解法和可视化。
复刻【我愿称之为写轮眼】，拿到一篇好文章，你能否看懂其精髓，能否用你自己能力把他拆解和复现出来。
类似音乐里面的扒谱，就是听到一首歌，你就知道在什么调、有哪些和弦，能把谱给扒出来，并且在其基础上升级改造。
再优秀的师傅也只能领你进门，关键时候点播你，不可能每时每刻手把手教你，其实真正的学习都是在你自己去玩、自己去拆解复现，培养自己的逻辑分析能力！！！
本质就是fast-follow，一是follow的了，你是不是能看懂，能不能用写轮眼复现出来；二是你有多快，兵贵神速，尤其是仿制药，me too药；
必备生物学知识
样本类型：
E3.5 ICMfromearly andmiddle blastocyst,
epiblast from E4.5 preimplantation embryos, （2i ESC）
E5.5 （EpiLCs）
E6.5 postimplantation embryos,
ectoderm dissected from E7.5 embryos
2i ESCs and EpiLCs, which resemble E4.5 epiblast and E5.5 epiblast
第一部分
these analyses suggest that we have identified bona fide真实的 SOX2 binding targets in early embryos
第一部分分析类似QC，说明你的数据是正常可信的。
PCA：The CUT&RUN data were reproduced between replicates
Peak annotation：Globally, 82.8 to 92.0% of SOX2 peaks occupied intergenic and intragenic regions away from promoters (fig. S2D), consistent with TFs predominantly binding to enhancers【我们的SOX9不是，很大一部分还是promoter】
KO or dTAG数据：We generated Sox2 maternal and zygotic knockout (mzKO) embryos and found that SOX2 binding was substantially diminished in mutant E3.5 ICM
motif enrichment：Across all stages, SOX2-bound putative enhancers were enriched for SOX2 motif and exhibited distinctmotif enrichment compared with assay for transposase-accessible chromatin sequencing (ATAC-seq)，which suggests that SOX2 binding is not simply dependent on chromatin accessibility【这个分析不错，TF binding不等于open chromatin】
初步整合binding和RNA：ICM-specific genes Upp1 and Spic exhibited SOX2-dependent transcriptional regulatory activity
Scatter plots showing global SOX2 binding correlation between consecutive stages.【一个非常流行的散点图，看相关性，也可以用热图代替】
Heatmaps showing the SOX2 binding signals and TF motif densities at stage-specific and shared distal SOX2 binding peaks.【聚类这个确实有点屌了，类似single-cell的marker】
第二部分
深入第一层生物学解读
These data demonstrate highly dynamic SOX2 binding during pluripotency progression
第二部分还是一个整体分析，已经开始进入biology了。
E3.5 ICM and E4.5 epiblast was highly divergent from that at other developmental stages 【PCA和相关性图说明】
E3.5 SOX2 binding sites preferentially harbored motifs of NR5A2, GATA, and TFAP2C, which were also enriched in accessible chromatin at the earlier stage [8-cell (8C)] but not the OCT4 and the composite OCT4-SOX2motifs (Fig. 1E).【binding和ATAC的motif enrichment】
These data suggest a poised state（开放预备态）of E3.5 ICM that coexpresses not only master pluripotency genes (Sox2, Oct4, and Nanog) but also PrE genes (Gata6) and early-stage TFs (Nr5a2 and Tfap2c)【已知了一些pluripotency、PrE、early的基因，motif富集、基因高表达，但我不懂poised state是如何推出来的？是不是得有binding+ATAC-的证据才行】
suggests a more gradual formative形成的-to-primed待发的 pluripotency transition. Because the pluripotency transition is a continuum, E5.5 epiblast may represent the formative state, whereas E6.5 epiblast may be transitionary between formative and primed pluripotency【主要是从PCA推理出来的】
A small number of binding sites were newly established in E7.5 ectoderm, accompanied by increased ATAC-seq and H3K27ac signals【同样是PCA推出，小整合分析】
Only a small subset (8.8%) of SOX2 distal peaks were shared among all seven cell types (Fig. 1G, “C8”).【这个peak marker heatmap真的很powerful】
第三部分
SOX2 is required for the ICM-TE lineage program in E3.5 ICM
正式进入生物学解读，开始有明确的假设：To investigate whether SOX2 binding in E3.5 ICM is linked to gene expression
其实也就是第二部分的subset分析，专注于E3.5和E4.5
图A的组合图真的是不错，多组学整合分析，binding和RNA的整合，heatmap聚类 + 累积分布 + 实例
然后做了单细胞测序（标准的WT vs SOX2 KO）
In total, 239 of the up-regulated genes were TE-enriched genes (such as Gata3, Krt8, Eomes, and Id2), whereas 99 of the down-regulated genes were ICMspecific (Fig. 2D and fig. S5C).【非常漂亮的富集证明SOX2确实是必须得】
Therefore, although the blastocyst morphology appears unaffected (10, 11), the ICM-TE transcription program is severely impaired in Sox2 mutant ICM.
The OCT4 and SOX2-OCT4 composite motif enrichment within SOX2 binding sites was lowin E3.5 ICM(Fig. 1E). They became enriched in E4.5 epiblast (Fig. 1G), which indicates that the cooperativity of these two pluripotency factors may only become mature when cells enter naive pluripotency.【就是第一部分的motif富集结果，可以看哪些其他TF与SOX2互作】
indicates that OCT4 and SOX2 have both shared and specific functions at this stage【一句永远都不太可能错的话，CNS顶刊尤其喜欢】
第四部分
改天再续，肯定会分析完的。