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Differentiation of PSC into lung organoids with endogenous functional alveolar macrophages and blood vessels

ABSTRACT: The goals of this study are to determine tissue composition of human lung organoids (hLOs) when maintained long-term (day 230). hLOs consist of alveolar epithelial cells, mesenchymal/endothelial cells, smooth muscle cells and immune cells. After dissociated hLOs, a target capture of 1x 104 cells was performed using the 10X Genomics Chromium Single Cell RNA sequencing. Briefly, single cell gel bead-in-emulsions(GEMs) are generated by passing cells with enzyme mix, partitioning oil, and barcoded gel beads by 10X Chromium chip. After GEM formation, the gel bead is dissolved and co-partitioned cell is lysed. Subsequently, reverse transcription (RT) occurs inside GEMs and barcoded full-length cDNA is generated. After RT, amplified cDNAs with barcode sequences (cell index and UMI) are pooled and single cell library is constructed using the Single Cell 3` Reagent Kit (v3 chemistry). The resulting library was sequenced on illumina HiseqX platform with 150bp paired end. Raw base calling files generated by illumine sequencing were demultiplexed based on the sample index read to generate FASTQ files using the 10X Genomics Cell Ranger (v3.0.2) pipeline. The raw reads were trimmed from the 3’ end to get the recommended number of cycle for read pairs (read1: 28bp; read2 : 91bp). The trimmed reads were mapped to the hg38 reference human genome with subsequent analysis, including filtering, barcode counting, and UMI counting. The resulting data were used to generate the multidimensional feature-barcode matrix using the CellRanger count with default parameters.

ORGANISM(S): Homo sapiens

PROVIDER: GSE142838 | GEO | 2020/01/03

REPOSITORIES: GEO

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Differentiation of PSC into lung organoids with endogenous functional alveolar macrophages and blood vessels

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