目前唯一一篇把单细胞测序应用到疾病发育上的高分paper，发表在了CSC上。
参考阅读：
首先明确一下概念：
帕金森疾病有一定的遗传因素，本文就是研究可遗传的这部分，GBA基因里的N370S突变会导致帕金森。The majority of PD cases are idiopathic, with only about 10% attributed to inherited PD cases
早期发现：increased ER stress, autophagic and lysosomal perturbations, and elevated a-synuclein release；内质网应激增加，自噬和溶酶体微扰增加，a-synuclein释放增加
基本的思路：
找出了一个核心的DEG：HDAC4
HDAC4靶向药物可以纠正帕金森症状
把核心DEG按照作用时间进行了分类。
根据临床信息把不同的疾病样本分层了stratify
HDAC4错误定位了 mislocalized
问题：
1. 如何找出HDAC4的？
这并不是一个转录因子
Pseudotime analysis of genes differentially expressed (DE) along this axis identified the transcriptional repressor histone deacetylase 4 (HDAC4) as an upstream regulator of disease progression.
HDAC4 was mislocalized to the nucleus in PD iPSC-derived dopamine neurons and repressed genes early in the disease axis, leading to late deficits in protein homeostasis.
HDAC4错误地进入了细胞核，抑制了那些早起基因，导致了蛋白质稳态缺陷。
2. 怎么找到靶向药物的？
3. 如何构建pseudotime的？
we identified a progressive axis of gene expression variation leading to endoplasmic reticulum stress.
Combining bulk and single-cell expression profiles, we identified a robust set of 60 genes whose expression captured an axis of variation between cells from controls and the remaining two PD GBA-N370S patients.
4. core gene set是怎么找出来的？
We next sought to identify a core set of genes consistently perturbed across both bulk RNA-seq and the single-cell transcriptomic signature. This core set was identified as the intersection of two gene sets from the analysis: (1) those DE in bulk RNAseq using DESeq2 after the removal of GBA3 (at 5% FDR) and (2) those DE across PC2 using switchde (at 5% FDR; Campbell and Yau, 2017; Figure S4B). We further refined this set to include only those additionally identified as discriminating marker genes after clustering the single-cell RNA-seq data using SC3 (Kiselev et al., 2017). By combining genes found through both bulk and single-cell DE as well as single-cell clustering (STAR Methods), we identified a core set of 60 genes, 52 of which were consistently downregulated and 8 of which were consistently upregulated in PD GBA-N370S iPSC-derived dopamine neurons (Figure S4B).
去文中一一找答案。。。
图1：
A：提纯purify iPSC-derived dopamine neurons，然后做bulk RNA-seq 和单细胞
B：increased expression of dopamine neuron marker genes多巴胺神经元的marker表达提升。感觉图表很业余，看得费力，大致就是想展现一些marker的表达情况，证明细胞是对的；
C：常规DEG的火山图，对比的bulk RNA-seq，我们不能用，因为样本不够纯；
D：常规的GO 注释图，down and up分类
图二：
A：单细胞常规的聚类分析，只不过用得是PC2和PC3，应该用了scater包，发现了分层现象，GBA3的表达变异被PC3捕获了；
B：常规的Over dispersion分析，鉴定出具有生物学差异的基因；才143个gene，有没有搞错；
C：an enrichment in the endoplasmic reticulum (ER) signal recognition particle (SRP) pathway in GBA3；ER通路在GBA3中高度富集；
D：挑了几个基因出来证明；
E：qRTPCR验证，靶向膜途径的SRP依赖性共翻译蛋白的升高对GBA3具有特异性，提出了本研究中使用的患者的分子分层，分子分层的依据；
The stratification of PSP from PD among these GBAN370S carriers by single-cell profiling of their iPSC-derived dopamine neurons is therefore consistent with the clinical stratification and reveals a potential disease-relevant pathway for PSP.
我们的这个项目适合分层吗？肯定可以
图三：
A：首先想办法构建了一个pseudo diease axis，然后判断core gene set在哪个位置发挥作用
B：统计学验证了core gene set的合理性；
C：挑了几个基因做了验证，DIV 22就是分化的天数，可以直观的看出基因到底是在哪一个区段开始发挥作用
<style></style> <style></style> <style></style> <style></style> <style></style> <style></style> <style></style>