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
What can microfluidics/microwells be combined with?
FACS in order to enrich the population
What are 2 considerations to take into consideration regarding the isolation of cells?
RNA can be lost or degraded during the process
if using frozen cells, information can be lost by altering the transcriptome
What is the best population type to enrich?
rare populations
How can we isolate WBC from RBC?
using FACS or column-based methods
What are the pros and cons to using FACS to isolate cells at a single level?
pros: sort cells based on phenotype using antibodies recognizing cell surface markers
cons: large amount of cells are needed, ells can be damages, velocity of sorting can damage them, cells can be lost during the process
What are the pros and cons to using Micromanipulation and LCM to isolate cells at a single level?
pros: cells can be isolated from fixed tissue and contain the topological information
cons: RNA can be degraded during the process, we have operator bias (each operator has to perform this isolation and micro-dissection since it is not autonomized), material can be lost and sample can be contaminated with nearby cells
What are the pros and cons to using Microfluids/microwells to isolate cells at a single level?
pros: highly standadized, automatized (less operator bias), small volumes, and there is visual confirmation
cons: loss of cells, low capture efficiency, not easy to use if cells are not in suspension, abundant population is needed
What is a velve-based microfluidic system?
system based on valves with 2 channels: one for cells and one for solution flow
one a single cell has been isolated, we can perform reverse transcriptase and cDNA amplification within a channel
What is the microwells system?
microwells confine cells for gravity and microwells can be sealed using a glass
this method allows us to isolate ells within each well that contains all the necessary reagents necessary for single cell RNA-seq
What is an emulsion-based/droplet approach?
cells are confined to droplets containing all the reagents required for single cell RNA-seq analysis
there are 3 inlets: one for oil (to create droplet), one for the cells, and one for the bead
What is InDrop?
an emulsion-based approach that is automatized
when pressure is applied, droplets are formed
UV light then allows the release of primers from hydrogels and lysis of cells occurs in the droplet
What are the pros and cons to using emusion-based/droplet approach (InDrop) to isolate cells at a single level?
pros: scalable, smaller volumes: better reproduction and higher detection
cons: high number of cell input and only 3’ mRNA information is obtained (not full-length)
After isolation, what must be done?
amplification of the cDNA or mRNA
Summarize the plate-based approaches:
FACS sorter can be used to isolate cells
microwells can be used to isolate cells at a single cell level
*allows us to profile a low number of cells
Summarize the pool-based approaches:
Drop-seq or InDrop: we start with a mass amount of cells and in the end we can profile about 500-2,000 cells
+allows us to profile a high number of cells
What method allows us to get information about the 3’ mRNA?
Tang method
What method allows us to get information about the full-length mRNA?
SMART-seq
What are barcodes or UMI important for?
to detect and quantify the number of transcripts (barcodes allow us to identify what kind of cell we are isolating)
What approach would we use to study gene expression?
full-length based approach or SMART-seq
What approach would we use to study a splicing variant or allelic variant?
single cell RNA-seq (individual cell approach)
Compare and contrast DropSeq and InDrops:
What is the best method in droplet-based approaches and why?
InDrops because it has a higher efficiency
What are the different parts that compose the oligo in droplet-based approach?
polidT: anneals with the polyA of the mRNA
UMI:barcode
R1 sequence: adaptor that performs the sequencing of the cDNA using NGS
What is PC1?
an advanced method of amplification of PCR
*can be used after R1 recognizes primer
What are the major differences in Dropseq and InDrop?
Dropseq uses an oligodT which includes the barcode and UMI so you have information on the quantification and the cell origin
SMARTseq lose information about the real quantification of mRNA and cell origin because the oligos do not contain UMI or barcodes
What does Dropseq, 10X, & InDrops amplify?
3’
What does SMARTseq amplify?
full length
What approach would we use to characterize the composition of tissues?
droplet-based approach
What approach would we use to characterize a rare population?
SMARTseq after enriching with FACS or a similar technique
What approach allows for the higher number of detection of genes?
SMARTseq
What approaches have the highest accuracy?
10X, Dropseq, & SMARTseq
What approach has the highest sensitivity?
SMARTseq because we can detect a low amount of mRNA while 10X sensitivity is lower
What is the best approach to use for rare cells?
SMARTseq because we are seaching for information on genes that have a low expression
What would we apply if we are looking for lineage tracing?
we would combine RNAseq with DNAseq or ATACseq or use spatial OMICS
Review the summary of the single cell RNA-seq methods:
