Lesson 25 Flashcards
what are the two main differences between smart-seq and drop-seq?
- 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
- 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
what do the single cell RNA protocols allow you to do?
dissect the population or tissue and study its response to different treatments to drugs
what do you use if you are interested in localizing your cells during disease progression?
spatial omics
what is the solution in order to bridge the gap between humans animal studies?
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
what are dendritic cells?
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
what are the two main populations of dendritic cells?
plasmacytoid DC (pDC) and the conventional DCs (cDC)
what is the marker for the identification of cDCs?
CD11c
what is a t-SNE plot?
a way to map and better visualize high dimensional data in a 2D plot, conserving the original dimensional structure of the population
how is it possible to see unbiased clusters when genes are expressed a high and low levels in a t-SNE plot?
DC subsets have well-defined expression signatures
AS DCs that Villani discovered contained what new markers?
AXL and SIGLE6
what is one of the main features to distinguish immune cells?
morphology
what is one important aspect of pDCs?
the production of an interferon
what cells could AS DC cells promote?
the proliferation of CD4 and CD8
what can steady state DC single cell data be used to map?
the ontogeny of disease-associated cells
what are the clinical signs of blastic plasmacytoid dendritic neoplasm (BPDCN)?
- 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
what are the four main functions of RNA-Seq?
- 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
what does scRNA-Seq give information about?
how disease cells are involved in a disorder → which cells are responsible for disease development, and how these cells are involved in disease progression
what is Alzheimer’s disease characterized by histologically?
by then parenchymal deposition of amyloid-beta Ab plaques
what is MARS-Seq?
a combination of pool-based and well-based approaches
LEARN MARS-Seq PROCESS
- 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. - Exonuclease I
- Sample pooling
- Second strain synthesis à the second molecule of RNA
can be produced in vitro - In vitro transcription
- DNase I
- RNA fragmentation
- RNA/ ssDNA ligation. Ligation is performed using a
sequence containing a plate barcode. - Reverse transcription à the cDNA is synthesized.
- Amplification of the cDNA
- Sequencing using specific primers
what other method besides MARS-Seq uses a t7 polymerase?
Tang / Surani
what are microglial cells?
a specialized population of macrophages involved in the depletion of damaged neurons and in the removal of infections
what did ScRNA-Seq discover associated with AD?
a unique microglial type - Diseased-Associated Microglial cells (DAM cells)
how are DAMs active in AD?
they display dynamics of activation during AD progression and change along the course of the disease
how do microglia present in AD?
microglia in the AD model display a transition from homeostatic microglia to DAM population as a function of disease progression
where are DAMs and AD plaques localized in the brain?
in the cortex
what did they use to assess the function of DAMs?
single molecule fluorescence n situ hybridization (smFISH) → an immunofluorescent based approach
DAM cells express genes involved in what pathways?
the phagocytic pathways and lipid metabolism
describe the phagocytic activity of DAMs:
they can phagocyte damaged neurons like microglial cells
what gene did they find in lipid metabolism and phagocytic DAM pathways?
Trem2
what are the three main microglial cell populations?
- homeostatic mocroglia
- Stage 1 DAMS: intermediate state
- Stage 2 DAMS: late / severe state → the frequency of DAM cells increase
what are DAMs activated by?
sequentially by Trem2-independent and dependent pathways
