But reading a few posts and issues here, it's not the way to go and I would like to understand why and to know how to do it properly. Compared with their circulating counterparts, tonsillar S+ and N+ Bm cells expressed, on average, more CD69, less Ki-67, reduced T-bet and several chemokine receptors differently (Fig. Asking for help, clarification, or responding to other answers. Cervia, C. et al. Does it look right? Neutrophils and emergency granulopoiesis drive immune suppression and As one can see in the pic below, the quality is quite different in each of the duplicated conditions. Tonsils were processed according to established protocols47,53. Academic theme for "~/Downloads/pbmc3k/filtered_gene_bc_matrices/hg19/", # Get cell and feature names, and total numbers, # Set identity classes to an existing column in meta data, # Subset Seurat object based on identity class, also see ?SubsetData, # Subset on the expression level of a gene/feature, # Subset on a value in the object meta data, # Downsample the number of cells per identity class, # View metadata data frame, stored in object@meta.data, # Retrieve specific values from the metadata, # Retrieve or set data in an expression matrix ('counts', 'data', and 'scale.data'), # Get cell embeddings and feature loadings, # FetchData can pull anything from expression matrices, cell embeddings, or metadata, # Dimensional reduction plot for PCA or tSNE, # Dimensional reduction plot, with cells colored by a quantitative feature, # Scatter plot across single cells, replaces GenePlot, # Scatter plot across individual features, repleaces CellPlot, # New things to try! Counts of SHM in S+ Bm cells remained high at month 12 (post-vaccination) compared with month 6 post-infection (pre-vaccination) (Fig. 4d). I hope it is useful. I know that I can do subsetting on just one gene in Seurat: However, I want to subset on multiple genes. B cell populations were identified using a WNN clustering and subsequent manual assignment. Differential gene expression identified higher expression of CR2, CD44, CCR6 and CD69 in tonsillar SWT+ Bm cells compared with blood SWT+ Bm cells, whereas the activation-related genes FGR and CD52 were higher in blood SWT+ Bm cells compared with their tonsillar counterparts (Extended Data Fig. Eg, the name of a gene, PC_1, a e, Presented are SHM counts in S+ Bm cells binding SWT, variant S (Sbeta and Sdelta) or RBD at month 6 (n=634 cells) and month 12 post-infection (n=197 cells; nonvaccinated); SHM counts in nave B cells (n=1,462) are shown as reference. These authors contributed equally: Yves Zurbuchen, Jan Michler. It would be nice if Satija lab could give more clear instruction on how to proceed in case of high versus low heterogeneity after subsettting. All plotting functions will return a ggplot2 plot by default, allowing easy customization with ggplot2. Most functions now take an assay parameter, but you can set a Default Assay to avoid repetitive statements. 351 2 15. At the transcriptional level, S+ Bm cells at month 6 post-infection upregulated genes associated with B cell activation and recent GC emigration35, such as NKFBIA, JUND, MAP3K8, CXCR4 and CD83, compared with S+ Bm cells at month 12 (Extended Data Fig. All samples were analyzed by flow cytometry and paired month 6 and 12 samples from nine patients also by single-cell RNA sequencing (scRNA-seq). Lines connect samples of same individual. If they had a confirmed SARS-CoV-2 infection and/or SARS-CoV-2 nucleocapsid-specific antibodies, they were considered SARS-CoV-2-recovered. ## [133] parallel_4.2.0 grid_4.2.0 tidyr_1.3.0 As you can see, many of the same genes are upregulated in both of these cell types and likely represent a conserved interferon response pathway. 6c). GOPB, Gene Ontology Biological Process. analyzed scRNA-seq data. I have added them all together and created the VlnPlot to check for the quality of the samples. How to create a virtual ISO file from /dev/sr0, Adding EV Charger (100A) in secondary panel (100A) fed off main (200A), English version of Russian proverb "The hedgehogs got pricked, cried, but continued to eat the cactus". Adv. 6, eabl9105 (2021). Below, we demonstrate methods for scRNA-seq integration as described in Stuart*, Butler* et al, 2019 to perform a comparative analysis of human immune cells (PBMC) in either a resting or interferon-stimulated state. column name in object@meta.data, etc. We longitudinally studied antigen-specific Bm cells in a cohort of 65 patients with COVID-19, 33 females and 32 males, including 42 with mild and 23 with severe disease course, during their acute SARS-CoV-2 infection and at months 6 and 12 post-infection. I have a few questions and was hoping you can help me address them; 5c). #2812 (comment). Atypical memory B cells are greatly expanded in individuals living in a malaria-endemic area. ## [1] systemfonts_1.0.4 sn_2.1.0 plyr_1.8.8 Zurbuchen, Y., Michler, J., Taeschler, P. et al. 2e), which correlated with an improved binding breadth, as measured by variant-binding ability of SWT+ Bm cells (Fig. ## Running under: Ubuntu 20.04.5 LTS First, we create a column in the meta.data slot to hold both the cell type and stimulation information and switch the current ident to that column. Since Seurat v3.0, weve made improvements to the Seurat object, and added new methods for user interaction. Well occasionally send you account related emails. Lines connect samples of same individual. Front Immunol. PubMed d, Heatmap displays V light (VL) gene usage in RBD+ and RBD Bm cells from scRNA-seq dataset of SARS-CoV-2-infected patients at month 6 and 12 post-infection. All samples were analyzed by flow cytometry and paired blood and tonsil samples from four patients also by scRNA-seq. c, Cohort overview of SARS-CoV-2 Vaccination Cohort. I have similar questions as @attal-kush with regards to reclustering of a subset of an integrated object. You can read more on the concept here in Martin's paper. By clicking Accept all cookies, you agree Stack Exchange can store cookies on your device and disclose information in accordance with our Cookie Policy. Also, cells previously occurring as cluster outliers from cl7 found their way to the corresponding clusters. 2.8 years ago. Gene set variation and enrichment analysis revealed a strong enrichment of a previously described B cell signature of IgDCD27CXCR5 atypical Bm cells from patients with systemic lupus erythematosus (SLE)36, in our SARS-CoV-2-specific CD21CD27FcRL5+ Bm cell subset (Fig. e, Circos plots of all persistent S+ Bm cell clones (left) and those adopting multiple Bm cell fates (right) are shown, with arrows connecting cells of months 6 with 12 and colored according to Bm cell phenotype at month 12. f, SHM counts were calculated in indicated S+ Bm cell subsets (unswitched, n=53; CD27lo resting, n=122; CD27hi resting, n=535; activated, n=713; CD21CD27FcRL5+, n=531). Multifactorial seroprofiling dissects the contribution of pre-existing human coronaviruses responses to SARS-CoV-2 immunity. Adding EV Charger (100A) in secondary panel (100A) fed off main (200A). b, Distribution of S+ Bm cell subsets is provided at month 6 preVac, month 12 nonVac and month 12 postVac.