Numbers
- Several hundred cell types in the human body
- Cell type specific genes (altered expression)
-
housekeeping genes are not altered between cell types (5000)
- constant between cell types
Bulk RNA-seq
- Condition tissue vs Control tissue
- determine difference in expression between two tissues (not cell specific)
- often not valid for individual cell types
Purified Bulk
-
attempt to isolate specific cell types before bulk sequencing
- Computational and Experimental purification (FACS antibodies/genetic drivers) methods
-
Pseudo-bulk takes single cell RNA-seq data and combines it so that it can be analyzed with classic bulk RNA-seq tools.
10X genomics scRNA=seq platform
-
droplets (aqueous in oil) are used to separate cells
- droplets are controlled so that each droplet contains one cell
- cells in droplets are given a barcode to allow for later identification
- cells can also have a sample id added
GEM (gel beads in emulsions)
Pseudo-time
- Inferring a process from snapshots
-
given discrete moments in time, how to determine continuous process from the points in time.
- Snapshots are transcriptomes at discrete time moments.
-
Snapshots can be taken from multiple cells (at different stages) at one time and arranged computationally
- An “asynchronous, heterogeneous moment”
Tools
- Seurat (R package)
- Monocle (R package)
example analysis flow
- filter cells
- group cells
- name cells
-
compare cells
- pseudo-bulk
- pseudo-time