Functional genomics Flashcards
What is the difference between a classical genetics approach and a functional genomics
approach?
Functional genetics is the study of how the genome (all genes) is related to the phenotype. Is a holistic approach. Has a high throughput.
Classical genomics is focused on single genes and has a low throughput.
What type of RNA is captured by polyT magnetic beads?
mRNA that contains a polyA tail.
What method would you use to measure accessibility of chromatin?
Using ATAC- sequencing (Assay for transposase accessible chromatin sequencing ) you can analyse chromatin accessibility. Key steps in this method are Cell dissociation –> Cell lysis –> Transposition –> Amplification
What is a ChIP-seq? What is meant by cross-linking and why is it necessary in ChIP-seq?
ChIP-seq Analyses protein–DNA interactions. It
Isolates a DNA sequence bound by specific proteins.
Cross linking is the linking of interacting DNA and protein. This is done so that it does not separate during immunoprecipitation, without this DNA-protein would seperate during purification.
What method would you use to measure TADs?
Hi-C which measures 3-dimensional structure of the chromosomes in the nucleus
What is Hi-C? Why does the DNA need to be biotinylated during Hi-C?
Hi-C measures 3-dimensional structure of the chromosomes in the nucleus
The biotin serves as a mark that can be used to purify fragments that are linked to a distantly
associated sequence. This saves on sequencing costs by removing unnecessary sequence.
What is the main sequence constraint for SpCas9 mediated gene editing?
The biotin serves as a mark that can be used to purify fragments that are linked to a distantly
associated sequence. This saves on sequencing costs by removing unnecessary sequence.
Why is it important to use a low lentivirus MOI in a CRISPR screen?
SpCas9 requires the presence of a protospacer adjacent motif (PAM) site next to the target
sequence. The PAM for SpCas9 is NGG.
Name three possible sources of input DNA for MPRAs.
De- Novo analysis: The sequences of intrest are synthesized.
Shearing: The genome is fragmented and sequences are randomly inserted into the MPRA vector.
PCR: Selected sequences are PCR amplified from the genome.
DNA capture: Antibodies are used to capture and pull down interactions and the DNA is inserted into the MPRA vector.
Why don’t you need a barcode for a STARR-seq experiment?
The sequence of interest is inserted into the 3’ UTR of the gene where it transactivates the
minimal promoter. The sequence is then incorporated into the mRNA and can be identified
directly by RNA-seq.
How does CRISPR work?
Decide which gene you want to cut. 2) Design a gRNA to target a specific PAM sequence near that region. 3) Express that gRNA in the cell of interest in addition to an endonuclease protein such as Cas9
