概要: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), may result in acute respiratory distress syndrome (ARDS), multi-organ failure and death. The alveolar epithelium is a major target of the virus, but representative models to study virus host interactions in more detail are currently lacking. Here, we describe a human 2D air-liquid interface culture system which was characterized by confocal-, electron-microscopy and single cell mRNA expression analysis. In this model, alveolar cells, but also basal cells and rare neuroendocrine cells, are grown from 3D self-renewing lung bud tip organoids. These cultures were readily infected by SARS-CoV-2 with mainly surfactant protein C-positive ATII-like cells being targeted. Consequently, significant viral titers were detected and mRNA expression analysis revealed induction of type I/III interferon response program. Treatment of these cultures with a low dose of interferon lambda dramatically reduced viral replication. Hence, these cultures represent an experimental model for SARS-CoV-2 infection and can be applied for drug screens.
项目整体设计: The researchers have setup a bronchioalveolar model for studying COVID-19. scRNAseq was performed to characterize the model on air-liquid interface bronchioalveolar cells not infected with SARS-CoV-2.
Bronchioalveolar cultures were grown from human lung bud tip organoids and differentiated at air-liquid interface in transwell inserts in the presence of canalicular stage fetal lung mesenchymal cells in the basal compartment
处理方案:
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提取方案:
Cultures were incubated with TrypLE express for 20 min at 37 C 5% CO2 and cells were dissociated by pipetting. Cells were washes once in cold PBS and resuspended in 200 L ice-cold PBS. Ice-cold methanol was added dropwise to the cell pellet for fixation. Cells were stored at -20 C until further processing. Single-cell mRNA sequencing was performed according to standard 10x Genomics 3 V3.1 chemistry protocol. Prior to loading the cells on the 10x Chromium controller, cells were rehydrated in rehydration buffer and counted to assess cell concentration. Cells were loaded and the resulting sequencing libraries were prepared following standard 10x Genomics protocol.
建库方案:
The DNA libraries was paired-end sequenced on an Illumina Novaseq S4, with a 2x150 bp Illumina kit.
测序信息
分子类型:
Poly(A)+ RNA
库的片段类型:
PAIRED
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测序平台:
ILLUMINA
测序仪型号:
Illumina NovaSeq 6000
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文章
An organoid-derived bronchioalveolar model for SARS-CoV-2 infection of human alveolar type II-like cells.
