1.Landscape of Infiltrating T Cells in Liver Cancer Revealed by Single-Cell Sequencing

2.Global characterization of T cells in non-small-cell lung cancer by single-cell sequencing

笔记点：

1. 是分别分选CD4、CD8和Treg送的SMART-seq2

2. We also filtered out low-quality cells following these steps: 1) to discard cells with the number of uniquely mapped read pairs less than 250,000; 2) to discard cells with RPKM of CD3D less than 1.0; 3) to discard FACS sorted CD8+ T cells with RPKM of CD8A less than 1.0 or with RPKM of CD4 larger than 10; 4) to discard FACS sorted CD4+ T cells with RPKM of CD4 less than 1.0 or with RPKM of CD8A larger than 10; 5) to discard cells that failed the outlier analysis.一种基于流式策略的单细胞质控方案。

   Screenshot 2022-03-07 at 10.55.30 PM.png
3. TCR analysis: workflow

4. 生信只是一种推断，验证终究还是湿实验

5. we filtered out TCR assemblies with alpha chain TPM < 10 or beta chain TPM < 15.

6. If the alpha chain of a cell was rearranged by the V segment of TRAV1-2 and one of the J segments TRAJ33, TRAJ20 and TRAJ12, it would be classified as a MAIT cell. If the α chain of one cell was rearranged
   by V segment TRAV10 and J segment TRAJ18, the cell was classified as an iNKT cell

7. 如何定义exhaustion\naiveness\cytotoxicity score？
   可以直接利用已有数据，严格分析耗竭细胞亚群和非耗竭细胞亚群的DEG，利用DEG进行评分（但注意，这个DEG CD4和CD8亚群是分开定义的）。也可以用常用的相关基因来进行定义，比如naive marker和cytotoxicity marker
   

   
   Monocle components were correlated with functional features of CD8+ T cells (the 3,700 cells as in Fig. 3b), including scores of exhaustion, cytotoxicity and naïveness calculated by the mean expression of gene sets related to these T cell status (see Methods). The solid lines represent LOESS fitting of the relationship between these scores with Monocle components. Violin plots in the top corners show the distribution of functional scores in various cell clusters, colored by means of the corresponding scores in each cluster, and black dots represent median values. P values were calculated by Pearson correlation, and P < 2.2 × 10-16 represents a P value approaching 0.

8. 人类T细胞的一些重要的marker

9. 这种生信做出来的伪流式图也挺有意思，有一点像文献精读1里面看到的metacell

   
   Intrinsic heterogeneity of suppressive tumor Tregs indicated by the bimodal distribution of TNFRSF9 expression. Cell density is color-coded, with red denoting high density and yellow low density. Such bimodal distribution of TNFRSF9 expression was not evident in other T cell clusters. The percentage and cell number of each subgroup are labeled in the corners.