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Regulation of Gene Expression > Ferrari & Lidonnici: Lecture XXV > Flashcards

Ferrari & Lidonnici: Lecture XXV Flashcards

Applying Single Cell Genomics to Research

1
Q

What are 2 single cell RNA-seq protocols we have seen?

A

Dropseq
SMARTseq

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2
Q

What do the single cell RNA-seq protocols do?

A

amplify mRNA transcript by using the Moloney Murine Leukemia Virus reverse transcriptase

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3
Q

What are the 2 main differences between Dropseq and SMARTseq?

A

Dropseq uses an oligo-dT to synthesize the 1st strand of cDNA, which includes a cellular barcode and the UMI sequence

SMARTseq does not use an oligio-dT

Dropseq only amplifies the 3’ end fragments using transposase Tn5

SMARTseq amplifies all the fragments

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4
Q

What do the single cell RNA-seq protocols allow us to do?

A

dissect the population or tissue, study the response to different treatments, and identify cells that weren’t identified previously that may be the main population responding to drug treatment

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5
Q

What can be learned from single cell?

A

taxonomy and census
A&P
pathology
normal physiological changes
developmental biology
molecular mechanisms

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6
Q

How can different immune cell types be identified?

A

combining different features: molecular markers, morphology, spatial localization, physical properties, developmental origins, transcriptional factor dependency, growth factor dependency, chromatin states, etc.

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7
Q

What are 2 population that make up dendritic cells?

A

plasmacytoid DCs (pDCs): involved in the production of interferon against viral infections

conventional DCs (cDCs)

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8
Q

What is the marker for the identification of conventional DCs?

A

CD11c

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9
Q

Review how to design an experiment using single cell approaches:

A
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10
Q

What does a tSNE plot useful for?

A

identify different clusters in an unbiased way

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11
Q

What subset has a well-defined gene expression signature?

A

dendritic cell subsets

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12
Q

What 2 DC subsets express high marker levels of CD11c?

A

DC2 & DC3

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13
Q

What marker does DC6 express?

A

CD141 and CD123 (also characterized by pDC)

this means DC6 is a pDC cluster

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14
Q

What is highly expressed in cluster DC3?

A

CD14

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15
Q

What is able to stimulate the proliferation of T cells?

A

CD1c+ of cluster DC2 and DC3

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16
Q

What is not able to induce T cell expansion?

A

pDCs

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17
Q

What does cluster 5 express high marker levels of?

A

AXL and SIGLEC6

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18
Q

What cells were called AS DCs?

A

cells that expressed the AXL and SIGLEC6 markers

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19
Q

What do AS DC subsets look like?

A

myeloid, not pDC

20
Q

What is an important aspect of pDC?

A

production of interferon

21
Q

What can pDC produce?

A

they are able to produce IFN

22
Q

What are AS DC able to do?

A

promote proliferation of both CD4 and CD8 T cells

23
Q

What is DC5?

A

new conventional DC subset from CD1C+ and CD141+ DC

24
Q

What does BPDCN stand for?

A

blastic plasmacytoid dendritic cell neoplasm

25
Q

What is blastic plasmacytoid dendritic cell neoplasm?

A

aggresssive hematological malignancy

rare phenomenom

predominantly affects males 3:1

clinical presentation: isolated cutaneous lesion

rapidly evolves (in months) to multiple sites: blood, bone marrow, lymph nodes, spleen, liver, CNS, lungs, kidneys and muscles

26
Q

What do BPDCN cells share a signature with?

A

pDC signature, and they have a distinct gene set

27
Q

List all the applications of single cell RNA-seq:

A

unbiased map of cell populations in healthy state

developing reagents and strategies for enrichment of specific populations

functional characterization in healthy state

mapping and studying disorders

28
Q

What is Alzheimer’s Disease from a histological point of view?

A

it is characterized by the parenchymal deposition of amyloid-beta (Ab) plates

29
Q

What is MARS-seq, and what was it used for?

A

a type of scRNA-seq that uses a combination of well-based and pool-based approaches

it was used to analyze the immune cells of AD patients

30
Q

Describe MARS-seq

A
  1. reverse transcription of the mRNA
  2. exonuclease I
  3. sample pooling
  4. second strain synthesis
  5. in vitro transcription
  6. DNAse I
  7. RNA fragmentation
  8. RNA/ssDNA ligation (ligation is performed using a plate barcode)
  9. reverse transcription → cDNA is synthesized
  10. amplification of cDNA
  11. sequencing using specific primers
31
Q

What did Tang and Surani (the 1st that ran scRNA-seq) use?

A

T7 polymerase

32
Q

What did scRNA-seq reveal in the AD experiment?

A

unique microglia type associated with AD

33
Q

What do the disease-associated microglia display during AD progression?

A

dynamics of activation

34
Q

What did the microglia in the AD model display?

A

transition from homeostatic microglia to DAM population as a function of disease progression

35
Q

What genes were enriched in the AD model population?

A

ApoE, Lpl, CD9, Trem2

36
Q

What is smFISH?

A

single molecule fluorescence in situ hybridization

used to identify the function of the cells in AD (or disease)

37
Q

What was identified in the AD model using smFISH?

A

DAM cells were near the β plaques

Csf1 and Cx3cr1 signals overlap

38
Q

What is Cx3cr1 a marker for?

A

homeostatic microglia (typically present in the brain)

39
Q

What is Csf1 a marker for?

A

DAMs

40
Q

What do DAM cells express?

A

genes found in the phagocytic pathway and in lipid metabolism

41
Q

What does the overlapping of Thioflavin-S and Lpl (marker for DAMs) indicate?

A

DAm cells have phagocytic activity (they can phagocyte damaged neurons like microglia cells)

42
Q

What was a gene found involved in AD in lipid metabolism and the phagocytic pathway?

A

Trem2

43
Q

What 3 microglia cell populations were found in the AD model?

A

homeostatic microglia
Stage 1 DAMs (intermediate state)
Stage 2 microglia (late/severe state) → DAM cells frequency increases

44
Q

What did scRNA-seq in the AD identify?

A

novel microglia type (DAM)

45
Q

What are DAMs?

A

AD-associated phagocytic cells conserved in mice and humans

associated sequentially by Trem2-independent and dependent pathways

46
Q

What can scRNA-seq be used for?

A

many different applications

Regulation of Gene Expression (28 decks)

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