RESUMO
Microdroplet single-cell ATAC-seq is widely used to measure chromatin accessibility, however, highly scalable and simple sample multiplexing procedures are not available. Here, we present a transposome-assisted single nucleus barcoding approach for ATAC-seq (SNuBar-ATAC) that utilizes a single oligonucleotide adaptor for multiplexing samples during the existing tagmentation step and does not require a pre-labeling procedure. The accuracy and scalability of SNuBar-ATAC was evaluated using cell line mixture experiments. We applied SNuBar-ATAC to investigate treatment-induced chromatin accessibility dynamics by multiplexing 28 mice with lung tumors that received different combinations of chemo, radiation, and targeted immunotherapy. We also applied SNuBar-ATAC to study spatial epigenetic heterogeneity by multiplexing 32 regions from a human breast tissue. Additionally, we show that SNuBar can multiplex single cell ATAC and RNA multiomic assays in cell lines and human breast tissue samples. Our data show that SNuBar is a highly accurate, easy-to-use, and scalable system for multiplexing scATAC-seq and scATAC and RNA co-assay experiments.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Heterogeneidade Genética , Neoplasias Pulmonares/metabolismo , Análise de Célula Única , Fatores de Transcrição/metabolismo , Animais , Antineoplásicos/farmacologia , Quimiorradioterapia , Cromatina/genética , Sequenciamento de Cromatina por Imunoprecipitação , Feminino , Humanos , Células K562 , Cinética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Masculino , Camundongos da Linhagem 129 , RNA-Seq , Dosagem Radioterapêutica , Fatores de Transcrição/genéticaRESUMO
Cellular reprogramming is driven by a defined set of transcription factors; however, the regulatory logic that underlies cell-type specification and diversification remains elusive. Single-cell RNA-seq provides unprecedented coverage to measure dynamic molecular changes at the single-cell resolution. Here, we multiplex and ectopically express 20 pro-neuronal transcription factors in human dermal fibroblasts and demonstrate a widespread diversification of neurons based on cell morphology and canonical neuronal marker expressions. Single-cell RNA-seq analysis reveals diverse and distinct neuronal subtypes, including reprogramming processes that strongly correlate with the developing brain. Gene mapping of 20 exogenous pro-neuronal transcription factors further unveiled key determinants responsible for neuronal lineage specification and a regulatory logic dictating neuronal diversification, including glutamatergic and cholinergic neurons. The multiplex scRNA-seq approach is a robust and scalable approach to elucidate lineage and cellular specification across various biological systems.