Ferrari & Lidonnici: Lecture XXV Flashcards
Applying Single Cell Genomics to Research
What are 2 single cell RNA-seq protocols we have seen?
Dropseq
SMARTseq
What do the single cell RNA-seq protocols do?
amplify mRNA transcript by using the Moloney Murine Leukemia Virus reverse transcriptase
What are the 2 main differences between Dropseq and SMARTseq?
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
What do the single cell RNA-seq protocols allow us to do?
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
What can be learned from single cell?
taxonomy and census
A&P
pathology
normal physiological changes
developmental biology
molecular mechanisms
How can different immune cell types be identified?
combining different features: molecular markers, morphology, spatial localization, physical properties, developmental origins, transcriptional factor dependency, growth factor dependency, chromatin states, etc.
What are 2 population that make up dendritic cells?
plasmacytoid DCs (pDCs): involved in the production of interferon against viral infections
conventional DCs (cDCs)
What is the marker for the identification of conventional DCs?
CD11c
Review how to design an experiment using single cell approaches:
What does a tSNE plot useful for?
identify different clusters in an unbiased way
What subset has a well-defined gene expression signature?
dendritic cell subsets
What 2 DC subsets express high marker levels of CD11c?
DC2 & DC3
What marker does DC6 express?
CD141 and CD123 (also characterized by pDC)
this means DC6 is a pDC cluster
What is highly expressed in cluster DC3?
CD14
What is able to stimulate the proliferation of T cells?
CD1c+ of cluster DC2 and DC3
What is not able to induce T cell expansion?
pDCs
What does cluster 5 express high marker levels of?
AXL and SIGLEC6
What cells were called AS DCs?
cells that expressed the AXL and SIGLEC6 markers
What do AS DC subsets look like?
myeloid, not pDC
What is an important aspect of pDC?
production of interferon
What can pDC produce?
they are able to produce IFN
What are AS DC able to do?
promote proliferation of both CD4 and CD8 T cells
What is DC5?
new conventional DC subset from CD1C+ and CD141+ DC
What does BPDCN stand for?
blastic plasmacytoid dendritic cell neoplasm
What is blastic plasmacytoid dendritic cell neoplasm?
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
What do BPDCN cells share a signature with?
pDC signature, and they have a distinct gene set
List all the applications of single cell 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 is Alzheimer’s Disease from a histological point of view?
it is characterized by the parenchymal deposition of amyloid-beta (Ab) plates
What is MARS-seq, and what was it used for?
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
Describe MARS-seq
- reverse transcription of the mRNA
- exonuclease I
- sample pooling
- second strain synthesis
- in vitro transcription
- DNAse I
- RNA fragmentation
- RNA/ssDNA ligation (ligation is performed using a plate barcode)
- reverse transcription → cDNA is synthesized
- amplification of cDNA
- sequencing using specific primers
What did Tang and Surani (the 1st that ran scRNA-seq) use?
T7 polymerase
What did scRNA-seq reveal in the AD experiment?
unique microglia type associated with AD
What do the disease-associated microglia display during AD progression?
dynamics of activation
What did the microglia in the AD model display?
transition from homeostatic microglia to DAM population as a function of disease progression
What genes were enriched in the AD model population?
ApoE, Lpl, CD9, Trem2
What is smFISH?
single molecule fluorescence in situ hybridization
used to identify the function of the cells in AD (or disease)
What was identified in the AD model using smFISH?
DAM cells were near the β plaques
Csf1 and Cx3cr1 signals overlap
What is Cx3cr1 a marker for?
homeostatic microglia (typically present in the brain)
What is Csf1 a marker for?
DAMs
What do DAM cells express?
genes found in the phagocytic pathway and in lipid metabolism
What does the overlapping of Thioflavin-S and Lpl (marker for DAMs) indicate?
DAm cells have phagocytic activity (they can phagocyte damaged neurons like microglia cells)
What was a gene found involved in AD in lipid metabolism and the phagocytic pathway?
Trem2
What 3 microglia cell populations were found in the AD model?
homeostatic microglia
Stage 1 DAMs (intermediate state)
Stage 2 microglia (late/severe state) → DAM cells frequency increases
What did scRNA-seq in the AD identify?
novel microglia type (DAM)
What are DAMs?
AD-associated phagocytic cells conserved in mice and humans
associated sequentially by Trem2-independent and dependent pathways
What can scRNA-seq be used for?
many different applications
