Ferrari: Lecture XXIV Flashcards
Applying Single Cell Genomics to Research
When did the idea of analyzing the genome at a single cell level arise?
1882
What are the 3 main factors that contribute to the importance of single cell analysis?
the advances in technologies all us to analyze the entire genome/transcriptome present in the cell
improvement of using instruments that allow us to sequence the DNA and RNA at a single cell level
the improvement on how to isolate cells at a single cell level
What can we explore with single cell analysis?
heterogenity within a tissue/bulk population
What can be discovered by analyzing DNA at a single cell level?
we can retrieve different mutations within the genome of cancer cells to study the progression of diseases
we can also describe the clonal structures to trace the evolution and spread of the disease
What do NGS platforms allow us to sequence?
DNA/RNA
What is the most important aspect in single cell analyzation?
quantification of RNA
What 2 methods can be used for the quantification of RNA?
full-length protocol (full-length mRNA)
tagged based protocols (only the 3’-5’ mRNA)
Why does RNA need to be amplified before being sequenced?
there is a very low amount of RNA in mammalians (0.1 pictograms)
What is the Tang/Surani method?
first method developed using T7 RNA polymerase
it is a form of reverse transcription
Describe the Tang/Surani method:
performs DNA amplification using in vitro transcription and it includes the use of an oligo-dT primer that anneals to the polyA of the mRNA
oligo-dT primer can synthesize the 1st strand of cDNA
the enzyme then adds polyA to the 5’ end of the cDNA
polyA allows another oligo to anneal, so the RNA polymerase can synthesize the 2nd strand
*using this approach, you get information only about the 3’ mRNA because the enzyme cannot synthesize the whole transcript
What is reverse transcription primarily carried out for?
create complementary DNAs (cDNAs) representing tissue- or cell-specific RNA populations
Review reverse transcriptase (Tang/Surani method):
https://www.thermofisher.com/it/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/rt-education/reverse-transcription-setup.html
What is SMARTseq?
a method that uses another enzyme: MMLV reverse transcriptase
What 2 activities does MMLV reverse transcriptase carry out?
template switching activity
terminal transferase activity
Describe SMARTseq:
oligoT (contains anchor sequence) anneals to the plyA of the mRNA and the enzyme can synthesize the 1st strand of cDNA
nt are added at the end (typically cytokines) that allows for the extension of cDNA and the annealing
another oligo containing polyG is added to polyC in order to perform the switching of the template
using the new oligo, it can synthesize the 2nd strand with the generation at the end of the full-length cDNA
What are unique molecular indexes (UMIs)?
sequences of 5 nt that improved single cell analysis
Why are UMIs important?
it allows us to quanitify the amount of transcript in the cell
What happens after the reverse transcription is performed using the barcoded oligodT (containing the UMI sequence)?
amplification of mRNA and at the end we have the total molecules of cDNA that correspond to that transcript
What is a protocol that can be used to isolate single cells?
FACS, which is done by isolating cells by staining them with antibodies that recognize surface markers
What is the droplet-based approach combined with?
laser microdissection
What does the droplet-based approach combined with laser microdissection allow?
allows us to isolate cells and have information about their localization in the tissue
What is an approach that uses the well-based platform?
microfluidics or micro wells
What is microfluidics/microwells?
cells are confined to fluidics or pipettes