Proliferation vs Differentiation in CD4 Th2 condition in vivo&vitro (PMID: 27176874)

Paper: Single-cell analysis of CD4+ T-cell differentiation reveals three major cell states and progressive acceleration of proliferation. Genome Biology. (2016)
Organism: Mus Musculus
Cell type: CD4 T-cell in vivo and vitro
Sample number: 287
Library preparation: Smarter/C1, 75PE

  1. mapped to Ensembl38.70 combined with ERCC / GSNAP / HTSeq / TPM / Poor quality cells are detected by mapping on 37 genes on the mitochondrial genome
  2. Clustering with dist@R, Functional Annotation with DAVID6.7, Cell to Cell correlation with Spearman
  3. CD3+CD4+CD62L- cells from mediastinal and mesenteric LNs of Nippostrongylus brasiliensis infected(5 days) mice (scRNAseq)
  4. Rank cells by expression of aggregated G2/M genes as cell cycle score (gene list is provided) (scRNAseq)
  5. Higher cell cycle links to IL-4 expression (scRNAseq)
  6. Th2 cells are tracked by Il13-eGFP mouse (bulk RNAseq)
  7. Cell cycle genes are upregulated from G0N to G2N(N=no IL13), more in G4P (bulk RNAseq)
  8. Select 46 cells from G0N, G2N, G4N, G4P, confirm 3 major states, G0N, G4P, G2n&G4N (scRNAseq)
  9. TFs analysis reveal the relationships between TF expression pattern and cell state (scRNAseq)
  10. Differentiated state(G4P) shows more homogenous (scRNAseq) :*because selected by IL13? (scRNAseq)
  11. Make model for T-cell behaviour at cellular level with ABC and death/remain identical / divide symmetrically
  12. FACS data, Image data analysis from culturing are also used for fitting and evaluation of the model
  13. From model, asymmetric division do not substantially contribute to Th2 differentiation
  14. From bulk time course data, ratio population from each state is predicted by specific gene expression profile from each state for validation (bulk RNAseq)
  15. PbTII CD4 T-cell (vs Plasmodium chabaudi AS) is transferred and recovered from spleen at days 2,3 and 4. scRNAseq with >1M / sample (scRNAseq)
  16. Increase in proliferation is observed in more mature, differentiated class in vivo (scRNAseq)
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