Discovery of 3 subpopulations in innate lymphoid cells 3 (PMID: 26878113)

Paper: The heterogeneity of human CD127(+) innate lymphoid cells revealed by single-cell RNA sequencing. Nature Immunology. (2016)
Organism: Homo sapiens
Cell types: Tonsil tissue from 3 adult patients with obstructive sleep apnea syndrome
Sample number: 648
Library preparation: Smart-seq2
DATA: GSE70580

  1. STAR v2.3.0 (default/only unique) / rpkmforgenes
  2. QC: RSeQC-2.3.4, TCR encoding genes are used to exclude T-cell contamination
  3. Batch effect is removed by COMBAT@SVA in bioconducotr
  4. t-SNE (Rtsne), clustering and bootstrapping with pvclust@CRAN.
  5. Biological variability is defined as those with higher variation than the spike-in RNAs
  6. SCDE for definition of cluster-specific genes.
  7. Lineage markers: T-cell(CD3,TCRαβ,TCRγδ), B-cell(CD19), monocytes/macrophages(CD14), DC(BDCA2,CD123,CD1a), mast cells(FcεRIα), progenitors(CD34)
  8. Cell sorting for ILCs: ILC1(Lin-CD127+NKG2A−CD16−CD117-CRTH2−), ILC2(Lin− CD127+NKG2A−CD16−CRTH2+), ILC3(Lin−CD45+CD127+CD161+CD117+CRTH2−)
  9. High variable trascriptomes in population with discrepancy between mRNA abundance and protein expression
  10. Average from as few as 10 cells provide good correlation between RNA vs Protein
  11. ILC3 consist of a) NKp44+ ILC3 cells(GM-CSF,TNF,IL-2(high),IL-22), b) CD62L+ ILC3 cells(GM-CSF,TNF,IL-2(low), unresponsive to IL-23+IL1b) and c) HLA-DR+ ILC3 cells(GM-CSF,TNF,IL-2)
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