RESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Single-nucleus RNA-seq (snRNA-seq) enables the interrogation of cellular states in complex tissues that are challenging to dissociate or are frozen, and opens the way to human genetics studies, clinical trials, and precise cell atlases of large organs. However, such applications are currently limited by batch effects, processing, and costs. Here, we present an approach for multiplexing snRNA-seq, using sample-barcoded antibodies to uniquely label nuclei from distinct samples. Comparing human brain cortex samples profiled with or without hashing antibodies, we demonstrate that nucleus hashing does not significantly alter recovered profiles. We develop DemuxEM, a computational tool that detects inter-sample multiplets and assigns singlets to their sample of origin, and validate its accuracy using sex-specific gene expression, species-mixing and natural genetic variation. Our approach will facilitate tissue atlases of isogenic model organisms or from multiple biopsies or longitudinal samples of one donor, and large-scale perturbation screens.
Assuntos
Anticorpos/análise , Núcleo Celular/genética , Genômica/métodos , Análise de Célula Única/métodos , Idoso , Idoso de 80 Anos ou mais , Animais , Núcleo Celular/química , Núcleo Celular/metabolismo , DNA/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/química , Neurônios/citologia , Neurônios/metabolismo , Córtex Pré-Frontal/química , Córtex Pré-Frontal/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Long-term hematopoietic stem cells (LT-HSCs) maintain hematopoietic output throughout an animal's lifespan. However, with age, the balance is disrupted, and LT-HSCs produce a myeloid-biased output, resulting in poor immune responses to infectious challenge and the development of myeloid leukemias. Here, we show that young and aged LT-HSCs respond differently to inflammatory stress, such that aged LT-HSCs produce a cell-intrinsic, myeloid-biased expression program. Using single-cell RNA sequencing (scRNA-seq), we identify a myeloid-biased subset within the LT-HSC population (mLT-HSCs) that is prevalent among aged LT-HSCs. We identify CD61 as a marker of mLT-HSCs and show that CD61-high LT-HSCs are uniquely primed to respond to acute inflammatory challenge. We predict that several transcription factors regulate the mLT-HSCs gene program and show that Klf5, Ikzf1, and Stat3 play an important role in age-related inflammatory myeloid bias. We have therefore identified and isolated an LT-HSC subset that regulates myeloid versus lymphoid balance under inflammatory challenge and with age.