Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

Regulation Of Gene Expression > Lesson 24 > Flashcards

Lesson 24 Flashcards

1
Q

what are the two most important parts of sinlge cel RNA Seq?

A

quantification of the RNA and the capture

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

what are the two methods of RNA quantification?

A

you can identify full length mRNA (full length protocol) or just the 3’-5’ mRNA (tagged base protocol)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

why must RNA be amplified using all transcriptome amplification?

A

this is necessary because in mammalian cells, the total amount of RNA is around 10 pictograms whereas mRNA is about 0.1 pictograms so it is a very small amount

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

what enzyme does the Tang/Surani method use?

A

T7 RNA polymerase

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

what is T7 RNA polymerase?

A

performs DNA amplification by using in vitro transcription and it includes the use of an oligo-dT primer that anneals the polyA of the mRNA (contains an adaptor sequence the allows the sequencing of the cDNA after the amplification of DNA)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

what information can you get from the Tang/Surani method?

A

only about the 3’mRNA because the enzyme cannot synthesize all the transcripts

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

what enzyme is used in SMART Seq?

A

MMLV reverse transcriptase

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

what are the two activities of MMLV reverse transcriptase?

A

template switching activity and terminal transferase activity

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

SMART-SEQ

A
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

what is the function of MMLV?

A

the enzyme is able to perform the stitching of the template and use the new oligo to synthesize the second strand at the end of the full length DNA

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

how big are unique molecular indexes?

A

5 nucleotides long

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

what do UMIs allows for?

A

the quantification of the real amount of transcripts preset in the cell

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

how does UMI work?

A

after the reverse transcription using these barcoded oligodT (containing the UMI sequence), there is the
amplification of our mRNA and at the end we have the total molecules of cDNA corresponding to that transcript. Using the UMI, we can quantify how many transcripts correspond to that gene

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

what is the easiest method of single cell isolation?

A

serial dilution of the sample in order to end up having only one cell in the total volume needed to perform the reaction

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

how do FACs work?

A

allow the isolation of cells by staining them with antibodies that recognize surface markers → FACs can distinguish the cells we want to isolate

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

what is the droplet based approach in single cell isolation?

A

we confine single cells in a droplet → but we lose the information about the localization of the cells in the tissue

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

what is later microdissection?

A

this approach allows to isolate cells and have information about their localization in the tissue, as by looking with the microscope during the dissection we know where the cells were localized and so after the harvesting and the sequencing we can go back to the tissue and rebuild where the cells with such profile where localized

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

what are some things we must consider in regards to preparation when analyzing isolated RNA?

A
  • in most applications the tissues must be fixed which can alter the transcriptome profile
  • if the tissues are frozen it can result in a loss of info from the transcriptome
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

what is the best thing to enrich?

A

rare populations

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

what are the pros and cons of FACS?

A

pros: we can sort cells based on the phenotype using antibodies recognizing cell surface markers

cons: we need a large amount of cells, and the method is not gentle (you can lose a lot of cells)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

what is micro manipulation and LCM?

A

under the microscope we can capture cells with a glass micropipette and aspirate them from the tissue

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

what are the pros and cons of micromanipulation?

A

pros: allows cells to be isolated from fixed tissues and to have topological info

cons: RNA can be degraded during this process so we have operator bias

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

describe the valve based microfluidic system:

A

system based on values: one is for cells and one is for the solution flow. A pressure is applied to the membrane of the valve, so they can block cells within the channel allowing the isolation of cells at the single cell level. In the channel there are all the reagent necessary for single cell RNA seq analysis, so once the single cell has been isolated, we can perform, within the channel, reverse transcription and cDNA amplification

24
Q

describe the microwell system:

A

the microwells basically confines cells for gravity; then we can seal microwells using a glass. In this way we can isolated cells within each well that contains all the reagents necessary for the single cell RNA seq

25
Q

what are the pros and cons of the microwell system?

A

pros: highly standardized and automatized

cons: loss of cells (pressure has to be used to confine cells in channels) and low capture efficiency

26
Q

describe the emulsion based droplet approach:

A

cells are confined in droplets contain all reagent required for the single cell RNA Seq analysis

27
Q

what is InDrop?

A

this is the typical emulsions based approach which is all automatized

28
Q

what is the commercial version of InDrop?

A

10x genomics

29
Q

what happens in InDrop analysis?

A

cells are confined thing the droplets together with reader sand with the hydrogel beads barcoded with the primer - once the droplets are generated UV light allows the release of the primers from the hydrogels → inside the droplets we have all the enzymes and primers, the reverse transcription can occur inside the droplet

30
Q

what are the pros and cons of InDrop?

A

pros: it is scalable

cons: we need a higher number of cells as input

31
Q

what are the two different approaches for amplifying cDNA or mRNA?

A

pooled based approach or individual cell amplification approach (plate based)

32
Q

how is the plate based approach used to amplify cDNA or RNA?

A

you can use FACs sorter to isolate cells and microwells to isolate them at the single cell level (allows you to profile a low number of cells)

33
Q

how is the pool based approach used to amplify cDNA or RNA?

A

Drop-See or InDrop → you start from a high number of cells

34
Q

using the Tang method, what type of information can you gain from sequencing?

A

only information about the 3’ end

35
Q

using the Smart Seq method, what type of information can you gain from sequencing?

A

get information about the full length of mRNA

36
Q

when sequencing, what two things can you use to tag sequences?

A

barcodes or unique molecular identifiers

37
Q

what do barcodes allow for?

A

allows you to identify what type of cell you are profiling

38
Q

what do unique molecular identifiers allow for?

A

allow you to detect and quantify the number of transcripts

39
Q

if you use barcodes in the retrotranscription step, what does this allow you to do?

A

profile cells in bulk → in the pull based approach you can use the cellular barcodes and the UMI to quantify the transcription for that cell and have the information for single cells

40
Q

what do you use if you have to study splicing variants or allelic variants?

A

single cell RNA Seq using the individual cell based approach

41
Q

what is the difference between InDrop and Drop Seq?

A

we have the same microfluidic approach with the 3 inlets: one for oil, one for reagents, and one for the beads → droplets are created containing the cell and all reagents (we have the lysis of the cell in the droplet and the hybridization of the primers with the mRNA) and the droplets are broken after the annealing of the mRNA with the primers conjugated with the beads generating STAMP

42
Q

what is STAMP generated in Drop Seq analysis?

A

single cell transcriptomes attached to Microparticles → reverse transcriptase with the template switching protocol occurs in pool

43
Q

what is the best method among the droplet based approaches?

A

InDrop

44
Q

how does DropSeq work?

A

cells are lysed → mRNA anneal to the oligo attached to the beads → the droplets are broken → there is a reverse transcription with the template switching → production of dsDNA that is now ready for the library preparation and sequencing

45
Q

how does InDrop work?

A

the reverse transcription occurs within the droplet and then there is the library preparation and sequencing

46
Q

what different parts make up an oligo?

A
  • polidT (that anneals with the poliA of the mRNA)
  • the UMI (barcode which allows us to have the information on the cell)
  • R1 sequence (an adaptor that allows us to perform the sequencing of the cDNA using NGS)
47
Q

following the lysis of the cell in InDrop, what is generated?

A

the cDNA is generated and amplified inside the droplet

48
Q

after the generation of dsDNA in InDrop, what occurs next?

A

it is ready for sequencing → the dsDNA is fragmented by using the transposes - cuts the product and during the cut adds a tag that contains another primer that allows us to sequence the product

49
Q

in InDrop, the primer used at the beginning contains a sequence (R1) that allows us to recognize a specific sequence - if we want to sequence this specific sequence, what other primer is also required?

A

PC1 - an advanced method of amplification of the PCR

50
Q

what is the first major difference between DropSeq and Smartse?

A

drop: uses an oligodT which includes the barcode and the UMI so you have the information about the quantification and cell origin
smart: we are losing the information about the real quantification of mRNA as well as the cell origin because the oligos do not contain cell barcodes

51
Q

what is the most important difference between dropseq and smartseq?

A

DropSeq = 3’

SmartSeq= full length

52
Q

why is so much information lost in DropSeq?

A

when we prepare the library for sequencing using a transposes, the transposes can fragment the cDNA and add the tag to different products, but only fragments that contain the 3’ end allowing you to have the information because the sequencing comes with the primer that anneals within the primers that were added to hydrogel and the other primers are in the tag added by the transposase → ALL OTHER FRAGMENTS ARE LOST

53
Q

what does the choice of platform depend on?

A

the biological question

54
Q

what allows for the detection of a higher number of genes compared to Drop Seq?

A

smart-seq → better sensitivity

55
Q

if you are interested in lineage tracking, what is the best method to use?

A

best to combine techniques → RNA-Seq with DNA-Seq / ATAC-Seq

Regulation Of Gene Expression (28 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions