Gene expression analysis and cell type identification were performed independently for villi and decidua samples using Seurat V2.0 (8). The gene expression matrix for each sample was generated, and ubiquitously expressed ribosomal protein–coding (RPS and RPL) and MALAT1 noncoding RNA genes were removed. Seurat objects were created for individual samples. Only those genes that were expressed in more than three cells and cells that expressed more than 100 genes were retained. All 10x Seurat objects for individual samples were merged into one 10x combined object (MergeSeurat), followed by scaling data (ScaleData) and finding variable genes (FindVariableGenes). All Drop-seq Seurat objects for individual samples were processed through similar steps as described above to generate a single Drop-seq combined object. Next, the union of the top 2000 variable genes for each, 10x and Drop-seq, combined objects was used to perform canonical correlation analysis (CCA) between 10x and Drop-seq datasets. Then, CCA subspaces were aligned using 1:16 CCA dimensions, which was followed by integrated t-SNE visualization for all cells. The expression of established lineage marker genes was used to assign cell types. Cells with >25% mitochondrial content (based on UMIs) was considered of poor quality and set aside in the second round of analysis. For villi, an average of 6% of cells in each lineage were poor-quality cells except for one cluster of cells comprising >50% poor-quality cells. This cluster also showed fewer UMIs than other lineages, was devoid of expression of cell type–specific genes, and therefore was removed in the second round of analysis. Next, we analyzed each individual cluster separately following similar strategy as described above. Individual cluster analysis identified ~4.5% cells to be doublets based on expression of multiple lineage markers that were removed in the final round of analysis. Subclustering analysis of only those clusters showing subpopulation was reported. In this process, we also identified proliferating subclusters within VCT, EB, FB1, and FB2 clusters of villi that showed expression of MKI67, TOP2A, TK1, and PCNA genes.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.