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

生物信息學

Cell theory:7個要點

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細胞異質性的緣由是morphology&genome&transcription&proteome，Cellar heterogeneity細胞異質性致使tumor。對於tumor使用PD1/PDL1無效，如今依據個性化須要使用single cell analysis，人羣隊列。

single cell analysis的挑戰有量小因此須要分離技術、須要的技術可重複。

中國湯富酬教授

 

Single cell isolation分離技術及各自特色

Limiting dilution將第一個孔內的細胞加入後一行細胞孔中，達到稀釋目的。

Micromanipulation用移液槍選出感興趣細胞

LCM熒光作selection。

FACS是流式細胞技術，如今主流技術，高通量分選。好比，10X genomics|Droplet

MACS是篩選加入磁珠的細胞。

10X genomics磁珠包裹在油滴裏面，特色是磁珠上的探針組成：R1+barcode（name）+UMI（identify different RNA sequence）+poly。

Droplet特色是液滴中包裹細胞。

 

Single cell cDNA amplification即cDNA擴增三種方式：

1.PCR擴增2.體外轉錄擴增3.phi29聚合酶擴增PMA

 

Milestone timeline

 

 

 

 

Analysis workflow：

 

 

 

 

Imputation：使用其餘細胞中的RNA彌補缺失值

降維方法：

PCA|tSNE|DM：

 

 

 

Analysis software：

Differential expression analysis

 

 

 

 

Cell subpopulation identification

 

 

 

 

 

細胞狀態軌跡和譜系cell trajectory/linage and pseudotime reconstruction/inference software:

 

 

 

Application好比胚胎髮育

 

 

 

Human cell atlas 人類細胞圖譜計劃：

Taxonomy:cell types細胞類型

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細胞內的 and inter-cellular circuit

 

 

Spatially resolved transcriptomes 空間轉錄：Spatially空間的

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

ST是將組織放在平鋪着的芯片上，看芯片對應孔的表達量，每個點表明取gene位置不一樣，可對應測同一個gene在不一樣位置上的表達量。

 

 

 

Slide-seq是將組織放在平鋪着的芯片上，看芯片對應孔的表達量，能夠作到單細胞水平。

LCM-seq

SeqFISH&MERFISH中每個點是一個細胞

 

 

 

Paired-cell sequencing針對肝臟細胞

Geo-seq|tomo-seq線性表達譜eg：將胚胎切成一段段，每一段的表達量

Spatial DB集合

空間轉錄組：每個細胞的空間信息和與環境的交互做用

 

LAMP webserver---Elementary knowledge網絡框架知識：

 

 

 

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