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Regulation Of Gene Expression > Lesson 25 > Flashcards

Lesson 25 Flashcards

1
Q

what are the two main differences between smart-seq and drop-seq?

A
  1. during the synthesis of the first strand of the cDNA, the Dropseq utilize an oligo- dT which includes a cellular barcode and the UMI sequence, whereas in the SMARTseq protocols the oligo-dT does not include these sequences
  2. during the amplification step in both protocols the
    cDNA molecules are fragmented to use the transposase TN5 but during this process in Dropseq protocol only 3’ end fragments are amplified in pull base approach, whereas in SMARTseq all the fragments are amplified, since the transposase add tag sequences at both ends of the fragments
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2
Q

what do the single cell RNA protocols allow you to do?

A

dissect the population or tissue and study its response to different treatments to drugs

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3
Q

what do you use if you are interested in localizing your cells during disease progression?

A

spatial omics

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4
Q

what is the solution in order to bridge the gap between humans animal studies?

A

to perform systematic single cell profiling of the main immune cell populations in order to identify these cell types and to define them from a molecular point of view

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5
Q

what are dendritic cells?

A

a specialized population the plays a role in pathogen sensing, antigen presentation, and T cell activation → monocytes are involved in phagocytosis, cytokine production, and macrophage source

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6
Q

what are the two main populations of dendritic cells?

A

plasmacytoid DC (pDC) and the conventional DCs (cDC)

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7
Q

what is the marker for the identification of cDCs?

A

CD11c

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8
Q

what is a t-SNE plot?

A

a way to map and better visualize high dimensional data in a 2D plot, conserving the original dimensional structure of the population

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9
Q

how is it possible to see unbiased clusters when genes are expressed a high and low levels in a t-SNE plot?

A

DC subsets have well-defined expression signatures

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10
Q

AS DCs that Villani discovered contained what new markers?

A

AXL and SIGLE6

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11
Q

what is one of the main features to distinguish immune cells?

A

morphology

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12
Q

what is one important aspect of pDCs?

A

the production of an interferon

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13
Q

what cells could AS DC cells promote?

A

the proliferation of CD4 and CD8

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14
Q

what can steady state DC single cell data be used to map?

A

the ontogeny of disease-associated cells

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15
Q

what are the clinical signs of blastic plasmacytoid dendritic neoplasm (BPDCN)?

A
  • aggressive hematological malignancy
  • rare
  • predominantly affects males
  • clinical presentation at time of diagnosis = isolated cutaneous lesions
  • rapidly evolves to multiple sites like the blood, bone marrow, lymph nodes, spleen liver, CNS, tonsils, lungs, kidneys, and muscles
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16
Q

what are the four main functions of RNA-Seq?

A
  1. Unbiased map of cell populations in healthy state
  2. Developing reagents and strategies for enrichment of
    specific populations
  3. Functional characterization in healthy state
  4. Mapping and studying disorders
17
Q

what does scRNA-Seq give information about?

A

how disease cells are involved in a disorder → which cells are responsible for disease development, and how these cells are involved in disease progression

18
Q

what is Alzheimer’s disease characterized by histologically?

A

by then parenchymal deposition of amyloid-beta Ab plaques

19
Q

what is MARS-Seq?

A

a combination of pool-based and well-based approaches

20
Q

LEARN MARS-Seq PROCESS

A
  1. Reverse transcription of the mRNAà they used
    primer containing UMI and cellular barcode (they
    cannot be used in Smart-Seq). After the first step of
    retro-transcription, the cells can be pooled together.
    Cellular barcodes are used to identify the different cell
    populations. UMI allows the quantification of RNA
    transcripts in the original cells. In the oligo there is also
    a T7 promoter because, instead of using Mo-MLV
    transcriptase, in this case they used the T7 transcriptase.
  2. Exonuclease I
  3. Sample pooling
  4. Second strain synthesis à the second molecule of RNA
    can be produced in vitro
  5. In vitro transcription
  6. DNase I
  7. RNA fragmentation
  8. RNA/ ssDNA ligation. Ligation is performed using a
    sequence containing a plate barcode.
  9. Reverse transcription à the cDNA is synthesized.
  10. Amplification of the cDNA
  11. Sequencing using specific primers
21
Q

what other method besides MARS-Seq uses a t7 polymerase?

A

Tang / Surani

22
Q

what are microglial cells?

A

a specialized population of macrophages involved in the depletion of damaged neurons and in the removal of infections

23
Q

what did ScRNA-Seq discover associated with AD?

A

a unique microglial type - Diseased-Associated Microglial cells (DAM cells)

24
Q

how are DAMs active in AD?

A

they display dynamics of activation during AD progression and change along the course of the disease

25
Q

how do microglia present in AD?

A

microglia in the AD model display a transition from homeostatic microglia to DAM population as a function of disease progression

26
Q

where are DAMs and AD plaques localized in the brain?

A

in the cortex

27
Q

what did they use to assess the function of DAMs?

A

single molecule fluorescence n situ hybridization (smFISH) → an immunofluorescent based approach

28
Q

DAM cells express genes involved in what pathways?

A

the phagocytic pathways and lipid metabolism

29
Q

describe the phagocytic activity of DAMs:

A

they can phagocyte damaged neurons like microglial cells

30
Q

what gene did they find in lipid metabolism and phagocytic DAM pathways?

A

Trem2

31
Q

what are the three main microglial cell populations?

A
  1. homeostatic mocroglia
  2. Stage 1 DAMS: intermediate state
  3. Stage 2 DAMS: late / severe state → the frequency of DAM cells increase
32
Q

what are DAMs activated by?

A

sequentially by Trem2-independent and dependent pathways

Regulation Of Gene Expression (28 decks)

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