Cell theory|Bulk RNA-seq|Cellar heterogeneity|Micromanipulation|Limiting dilution|LCM|FACS|MACS|Drop

生物信息学

Cell theory:7个要点

All known living things are made up of one or more cells.

All living cells arise from pre-existing cells by division.

The cell is the fundamental unit of structure and function in all living organisms.

The activity of an organism depends on the total activity of independent cells.

Energy flow (metabolism and biochemistry) occurs within cells.

Cells contain DNA which is found specifically in the chromosome and RNA found in the cell nucleus and cytoplasm.

All cells are basically the same in chemical composition in organisms of similar species.

 

Genotype-Transcription-gene function phenotype

Bulk RNA-seqhttps://www.youtube.com/watch?v=UgslMZ74ums

Cellar heterogeneity细胞异质性的缘由是morphology&genome&transcription&proteome，Cellar heterogeneity细胞异质性致使tumor。对于tumor使用PD1/PDL1无效，如今依据个性化须要使用single cell analysis，人群队列。

single cell analysis的挑战有量小因此须要分离技术、须要的技术可重复。

中国汤富酬教授

 

Single cell isolation分离技术及各自特色

Limiting dilution将第一个孔内的细胞加入后一行细胞孔中，达到稀释目的。

Micromanipulation用移液枪选出感兴趣细胞

LCM荧光作selection。

FACS是流式细胞技术，如今主流技术，高通量分选。好比，10X genomics|Droplet

MACS是筛选加入磁珠的细胞。

10X genomics磁珠包裹在油滴里面，特色是磁珠上的探针组成：R1+barcode（name）+UMI（identify different RNA sequence）+poly。

Droplet特色是液滴中包裹细胞。

 

Single cell cDNA amplification即cDNA扩增三种方式：

1.PCR扩增2.体外转录扩增3.phi29聚合酶扩增PMA

 

Milestone timeline

 

 

 

 

Analysis workflow：

 

 

 

 

Imputation：使用其余细胞中的RNA弥补缺失值

降维方法：

PCA|tSNE|DM：

 

 

 

Analysis software：

Differential expression analysis

 

 

 

 

Cell subpopulation identification

 

 

 

 

 

细胞状态轨迹和谱系cell trajectory/linage and pseudotime reconstruction/inference software:

 

 

 

Application好比胚胎发育

 

 

 

Human cell atlas 人类细胞图谱计划：

Taxonomy:cell types细胞类型

Histology：cell neighborhood and position

Development ：transitions to differentiated cell types

Physiology and homeosrasis同源性：cycles，transient responses and plastic state

Disease：cells and cellular ecosystem

Molecular mechanism：intracellular细胞内的 and inter-cellular circuit

 

 

Spatially resolved transcriptomes 空间转录：Spatially空间的

网址：https://www.spatialomics.org/SpatialDB/

ST是将组织放在平铺着的芯片上，看芯片对应孔的表达量，每个点表明取gene位置不一样，可对应测同一个gene在不一样位置上的表达量。

 

 

 

Slide-seq是将组织放在平铺着的芯片上，看芯片对应孔的表达量，能够作到单细胞水平。

LCM-seq

SeqFISH&MERFISH中每个点是一个细胞

 

 

 

Paired-cell sequencing针对肝脏细胞

Geo-seq|tomo-seq线性表达谱eg：将胚胎切成一段段，每一段的表达量

Spatial DB集合

空间转录组：每个细胞的空间信息和与环境的交互做用

 

LAMP webserver---Elementary knowledge网络框架知识：

 

 

 

Mooc：w3cschool网址：https://www.w3cschool.cn/

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