RESUMO
Large-scale surveys of single-cell gene expression have the potential to reveal rare cell populations and lineage relationships but require efficient methods for cell capture and mRNA sequencing. Although cellular barcoding strategies allow parallel sequencing of single cells at ultra-low depths, the limitations of shallow sequencing have not been investigated directly. By capturing 301 single cells from 11 populations using microfluidics and analyzing single-cell transcriptomes across downsampled sequencing depths, we demonstrate that shallow single-cell mRNA sequencing (~50,000 reads per cell) is sufficient for unbiased cell-type classification and biomarker identification. In the developing cortex, we identify diverse cell types, including multiple progenitor and neuronal subtypes, and we identify EGR1 and FOS as previously unreported candidate targets of Notch signaling in human but not mouse radial glia. Our strategy establishes an efficient method for unbiased analysis and comparison of cell populations from heterogeneous tissue by microfluidic single-cell capture and low-coverage sequencing of many cells.
Assuntos
Córtex Cerebral/crescimento & desenvolvimento , Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , RNA Mensageiro/análise , Análise de Sequência de RNA/métodos , Transdução de Sinais/genética , Animais , Córtex Cerebral/metabolismo , Desenho de Equipamento , Humanos , Camundongos , Técnicas Analíticas Microfluídicas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/fisiologiaRESUMO
The Staphylococcus aureus copA gene codes for a putative copper-translocating P-type ATPase and the downstream copZ gene codes for a copper chaperone. Genome database analyses demonstrate that these copper transport genes are highly conserved in S. aureus. The expression of copA and copZ was inducible by copper and to some extent by ferric and lead ions. A mutant strain containing a partially deleted copA gene was more sensitive than the parent strain to copper, ferric and lead ions. The copper-sensitive phenotype was due to the accumulation of intracellular copper and thus the copA product is involved in the export of copper ions. The metal-sensitive phenotype of the mutant was complemented in trans by a 2.7 kbp DNA containing copA. We have cloned and overexpressed the metal-binding domains of CopA and CopZ and have shown by site-directed mutagenesis that the cysteine residues in the CXXC metal-binding motif in CopA are involved in copper binding and thus play an important role in copper transport in S. aureus.
