李寅青 yinqingl@tsinghua.edu.cn 药学院，生物医学馆B405
what is a brain made of:
- the arrangement?
- different types of cells
GFP染色、Glogi染色
Brain is made of vastly diverse cell types
Transcriptome provides a unified view of cell classification
- Morphology
- Spatial organization
- Electrophysiology
这些差异都与基因表达pattern的差异有关。signaling/patterning/channels genes
How to obtain transcriptome:
- cDNA microarray, 1995
- whole genome genome expression profiling using tiling array, (RNA-scope), Affymetrix, 2002
- mRNA-seq: direct sequencing of mRNAs using next generation sequencing techniques (NGS)
Sequencing technology: price
Bridge sequencing
library preparation ⇒ cluster growth (single molecule array) ⇒ bridge amplification ⇒ linearization ⇒ sequencing ⇒ image acquisition ⇒ base calling
sequencing:
- 去除negative primers
- synthesis (SBS) close-up: 每一轮荧光分子发光。
mapping RNA seq reads
counting gene expression: generate possible pairings of genes; align reads to possible junctions.
单细胞测序
Tissues are heterogeneous
- 组织消化：化学消化、生物消化
- 癌组织消化最容易测到的就是免疫细胞。
Single cell RNA sequencing workflow
- single cell RNA-seq
- expression profile
- clustering: the expression level distribution across different types of cells in a region
single cell collection：
- 通量比较低：
- limiting dilution
- micromanipulator
- microfluidics
- FACS
- LCM
- 基于微流控制：
- 液滴高通量单细胞测序（几千-一万），drop-seq, inDrop
- 高通量单细胞测序（百万级），10xGenomics, SPLit-seq
- methanol fixed cells or extracted nuclei，防止NA漏出细胞
- 不断dilute & distribute：百万级铺板⇒取一个孔铺板⇒取一个孔铺板⇒取一个孔铺板，大概率四次后百万级的细胞数量会变成一个孔一个细胞。
Clustering analysis
- Principle Component Analysis (PCA)：寻找相关性
- 信息熵
Identification of cell heterogeneity
- 对组织做单细胞测序
- 聚类分析
大脑cells的难点在于细胞都交织在一起。
- 原位取细胞：速度慢，难度高。
- 固定细胞核，把其他部分打碎，对细胞核进行测序。细胞核代表整个细胞。
Single nuclei RNA sequencing
- 细胞核与整个细胞表达谱的相关性比较高。
但是，single cell transcriptional profiling is noisy
- Traditional PCA doesn’t work.
- noise
- dropout noise
- sampling noise
- how to differentiate marker gene and other genes
- expand data: use more cells
- nonlinear transformation
- Bi-clustering on stochastic neighbor embedding
- 引入原位组织染色图片上细胞的分布。通过如何的transformation将空间分布与测序结果对应起来（suppose 空间上接近的细胞其基因表达类似。）
Major cell types in mouse hippocampus.
核酸类似物+荧光标签，流式分选，区分出新生的细胞。Dynamic gene expression along maturation
Single nuclei RNAseq of enriched rare cells enables discovery of GABAergic neurogenesis in spinal cord.
Multitude of cellular identity
- 多维的信息，不只有转录组。
- 因此出现spatial transcriptome
single-cell RNAseq + multiplexed Error Robust Fluorescence in situ hybridization (MERFISH)
⇒
- spatial organization of cells in the preoptic region
- male and female social behaviors parenting
如果在原位进行测序，而不用杂交探针之类的？
- 原位核酸扩增
- 原位水凝胶固定，3D cDNA library
- spatial cell typing，3D imaging，只需要测几十个bp就可以鉴定gene
另外一个原位测序思路
- DNA patterned chip
- CID sequencing
- Stereo-seq capture chip
- in situ mRNA capture
- Library construction & sequencing
- Spatially resolved transcriptomic profiling