Week 10 Flashcards
How to make a non-homologous end rejoining (NHEJ)?
Double DNA break leads to gene inactivation –> miRNA site inactivation.
Can lead to frameshift.
What is base editing?
Deaminating and replacing specific base. Efficient and not often naturally repaired.
Whole genome library screening
Guide RNA mixed with cas9. Cut and inactivate gene. In each cell, only 1 specific gene will be inactivated. If crucial, cell dies.
Transcriptomics disadvantages
Need to design primer specific for each qPCR (quantitative pcr)
How microarray works?
CDNA probes attached to wells —> hybridise w control and non-control cells
But limited by probe making
Next generation sequencing
Incorporate cDNA into sequence of full cell. Primers are oligonucleotide based and laser detects their incorporation. Lysis —> library
Why use single nucleotide sequencing?
Bulk can mask cellular heterogeneity —> averaging artifacts
Split pool/ combinational in situ barcoding sequencing
Fix many cells into single well then split to analyse.
Nanodroplet sequencing method
Cell encapsulated into droplet —> lyse —> mRNA bind to oligoDT probes —> RNA hybridisation —> reverse transcription
Batch effect causes
Technical variability, changes in sample/processing, library prep/sequencing tech
Biological variability
Patient, environment, evolution
Validation methods
Antibody immunostaining
Mass spectrometry
Transgene knockout
Pseudotemporal ordering
Look at cells and infer a “life trajectory”. Can see them as “fake timepoints” at the same time.
Cell atlasing
Take tissue apart and look at markers. Can know processes of tissues
Comparative analysis in single cell RNA sequencing (scRNQseq)?
Can identify if a subpopulation of rnas are associated with disease
