From 39d0c4d5ef3c283ca0863cb6fccebb8d5e8b6f8b Mon Sep 17 00:00:00 2001 From: Dan Skelly Date: Thu, 17 Oct 2024 02:19:44 -0400 Subject: [PATCH] correct KH-doublet and c15 vs c11 confusion --- _episodes_rmd/06-Biology-Driven-Analyses.Rmd | 19 ++++++++++++------- 1 file changed, 12 insertions(+), 7 deletions(-) diff --git a/_episodes_rmd/06-Biology-Driven-Analyses.Rmd b/_episodes_rmd/06-Biology-Driven-Analyses.Rmd index 9b0d56f..2288d29 100644 --- a/_episodes_rmd/06-Biology-Driven-Analyses.Rmd +++ b/_episodes_rmd/06-Biology-Driven-Analyses.Rmd @@ -544,22 +544,25 @@ This looks better -- the markers are more specific. In this violin plot we do have some instances where a marker seems to be specific to two or three cell -clusters (e.g. Vsig4). +clusters (e.g. Lyve1). -Let's try to figure out what is going on with Vsig4: +In an older version of Seurat the gene Vsig4 was +also picked out. Let's try to figure out what is going on with Vsig4: ```{r vsig4} FeaturePlot(liver, "Vsig4", cols = c('lightgrey', 'red'), label = TRUE, label.size = 6) ``` -This is marking clusters c3 and c15. -Clusters c3 and c8 are very near each other. Vsig4 is an immune +This is marking clusters c3 and a small cluster +that may be labeled as c15 +(or may be part of c11). +Vsig4 is an immune protein (V-set and immunoglobulin domain containing 4). The protein [is expressed](https://www.proteinatlas.org/ENSG00000155659-VSIG4/tissue) selectively in -- among other cell types -- Kupffer cells, -which are the resident macrophages in the liver. Clusters c3 and c8 may be +which are the resident macrophages in the liver. Cluster c3 may be Kupffer cells. Let's check a famous macrophage marker, F4/80 (gene name Adgre1): @@ -568,14 +571,16 @@ FeaturePlot(liver, "Adgre1", cols = c('lightgrey', 'red'), label = TRUE, label.size = 6) ``` -Cluster c15 expresses *Adgre1* but is near the hepatocyte cluster +Cluster c15 (if it exists) +expresses *Adgre1* but is near the hepatocyte cluster we just discussed. In fact it is located between the hepatocyte and Kupffer cell clusters. Cluster c15 might represent hepatocyte-Kupffer cell doublets. Consistent with this theory, cluster c15 has intermediate expression of Kupffer cell-specific *Adgre1* and hepatocyte-specific *Fabp1*. ```{r doublets, fig.width = 8, fig.height = 4} -VlnPlot(liver, c("Adgre1", "Fabp1"), idents = c('c3', 'c15', 'c1'), sort = T) +VlnPlot(liver, c("Adgre1", "Fabp1"), idents = c('c3', 'c15', 'c11', 'c1'), + sort = T, pt.size = 0) ``` Let's store our labels and look at what remains unidentified.