This script is used for analysis of selected subsets/clusters after running Two_DRG_sample_scRNAseq_analysis

load libraries

# setwd("D:/Bioinformatics Projects/DRG")
library(Seurat)
library(dplyr)
library(cowplot)
library(ggplot2)
library(rstatix)
library(SingleCellExperiment)
library(ComplexHeatmap)
library(RColorBrewer)
library(circlize)
library(patchwork)

Read the subset .RDS file

Re-scale, SCTransform and find variable genes

Dimplot of subset, on tsne and umap embeddings

tsne and umap by groups

find markers for each cluster, and write top markers to .csv file

violin plot of top marker for each cluster

Top ten markers are

Cell numbers for each cluster are 1163, 877, 844, 762, 731, 650, 650, 554, 538, 529, 499, 450, 429, 208, 192

Total number of cels for all clusters is: 9076

define genes of interest

Geneset #1: Harrison pick: Prlr, Prl, Oprm1, Oprk1, Oprd1, Penk, Pdyn, Pomc, Adra2a, Adrb1, Adrb2, Htr1b, Htr1d, Scn10a, Trpv1, Trpa1, Trpm8, Piezo1, Piezo2, Calca
Geneset #2: SQ pick: Trpm8, Tac1, Nefh, Calca, Piezo2, Pvalb, Calcb, Adcyap1, Ntrk1, Ntrk3, Scn1a, Scn1b, Scn11a, S1pr3, Osmr, Nppb, Il31ra, Mrgprd, Sst, Jak1, Trpc3
Geneset #3: Allen brain SMARTseq 2: Snap25, Gad1, Gad2, Slc32a1, Slc17a7, Lamp5, Aqp4, Gfap, Ndnf, Sncg, Vip, Trem2, Sst, Pvalb, Cux2, Rorb, Fezf2, Foxp2, Npas4, Mbp, Cldn5, Ctss, C1qa, Map2k1, Mapk1, Mapk3

Heatmap of selected genes:

Harrison picked genes

SQ picked genes

Allen Brain Marker genes

Violin plot of selected genes

3D visualization of UMAP of this subset

Here is a 3D visualization of this subset of cells:
3D UMAP embeddings

Using Shiny to visualize

session info

Print session information

## R version 4.2.0 (2022-04-22 ucrt)
## Platform: x86_64-w64-mingw32/x64 (64-bit)
## Running under: Windows 10 x64 (build 19044)
## 
## Matrix products: default
## 
## locale:
## [1] LC_COLLATE=English_United States.utf8 
## [2] LC_CTYPE=English_United States.utf8   
## [3] LC_MONETARY=English_United States.utf8
## [4] LC_NUMERIC=C                          
## [5] LC_TIME=English_United States.utf8    
## 
## attached base packages:
## [1] grid      stats4    stats     graphics  grDevices utils     datasets 
## [8] methods   base     
## 
## other attached packages:
##  [1] patchwork_1.1.1             circlize_0.4.15            
##  [3] RColorBrewer_1.1-3          ComplexHeatmap_2.12.1      
##  [5] SingleCellExperiment_1.18.0 SummarizedExperiment_1.26.1
##  [7] Biobase_2.56.0              GenomicRanges_1.48.0       
##  [9] GenomeInfoDb_1.32.2         IRanges_2.30.0             
## [11] S4Vectors_0.34.0            BiocGenerics_0.42.0        
## [13] MatrixGenerics_1.8.1        matrixStats_0.62.0         
## [15] rstatix_0.7.0               ggplot2_3.3.6              
## [17] cowplot_1.1.1               dplyr_1.0.9                
## [19] SeuratObject_4.1.3          Seurat_4.3.0               
## 
## loaded via a namespace (and not attached):
##   [1] backports_1.4.1        plyr_1.8.7             igraph_1.3.2          
##   [4] lazyeval_0.2.2         sp_1.5-0               splines_4.2.0         
##   [7] listenv_0.8.0          scattermore_0.8        digest_0.6.29         
##  [10] foreach_1.5.2          htmltools_0.5.2        fansi_1.0.3           
##  [13] magrittr_2.0.3         doParallel_1.0.17      tensor_1.5            
##  [16] cluster_2.1.3          ROCR_1.0-11            limma_3.52.2          
##  [19] globals_0.15.1         spatstat.sparse_3.0-0  colorspace_2.0-3      
##  [22] ggrepel_0.9.1          xfun_0.31              crayon_1.5.1          
##  [25] RCurl_1.98-1.6         jsonlite_1.8.0         progressr_0.10.1      
##  [28] spatstat.data_3.0-0    iterators_1.0.14       survival_3.3-1        
##  [31] zoo_1.8-10             glue_1.6.2             polyclip_1.10-0       
##  [34] gtable_0.3.0           zlibbioc_1.42.0        XVector_0.36.0        
##  [37] leiden_0.4.2           GetoptLong_1.0.5       DelayedArray_0.22.0   
##  [40] car_3.1-0              shape_1.4.6            future.apply_1.9.0    
##  [43] abind_1.4-5            scales_1.2.0           DBI_1.1.3             
##  [46] spatstat.random_3.0-1  miniUI_0.1.1.1         Rcpp_1.0.8.3          
##  [49] viridisLite_0.4.0      xtable_1.8-4           clue_0.3-62           
##  [52] reticulate_1.25        htmlwidgets_1.5.4      httr_1.4.3            
##  [55] ellipsis_0.3.2         ica_1.0-2              farver_2.1.0          
##  [58] pkgconfig_2.0.3        sass_0.4.1             uwot_0.1.14           
##  [61] deldir_1.0-6           utf8_1.2.2             labeling_0.4.2        
##  [64] tidyselect_1.1.2       rlang_1.0.3            reshape2_1.4.4        
##  [67] later_1.3.0            munsell_0.5.0          tools_4.2.0           
##  [70] cli_3.3.0              generics_0.1.2         broom_1.0.0           
##  [73] ggridges_0.5.3         evaluate_0.15          stringr_1.4.0         
##  [76] fastmap_1.1.0          yaml_2.3.5             goftest_1.2-3         
##  [79] knitr_1.39             fitdistrplus_1.1-8     purrr_0.3.4           
##  [82] RANN_2.6.1             pbapply_1.5-0          future_1.26.1         
##  [85] nlme_3.1-157           mime_0.12              ggrastr_1.0.1         
##  [88] compiler_4.2.0         rstudioapi_0.13        beeswarm_0.4.0        
##  [91] plotly_4.10.0          png_0.1-7              spatstat.utils_3.0-1  
##  [94] tibble_3.1.7           bslib_0.3.1            stringi_1.7.6         
##  [97] highr_0.9              lattice_0.20-45        Matrix_1.5-3          
## [100] vctrs_0.4.1            pillar_1.7.0           lifecycle_1.0.1       
## [103] GlobalOptions_0.1.2    spatstat.geom_3.0-3    lmtest_0.9-40         
## [106] jquerylib_0.1.4        RcppAnnoy_0.0.19       data.table_1.14.2     
## [109] bitops_1.0-7           irlba_2.3.5            httpuv_1.6.5          
## [112] R6_2.5.1               promises_1.2.0.1       KernSmooth_2.23-20    
## [115] gridExtra_2.3          vipor_0.4.5            parallelly_1.32.0     
## [118] codetools_0.2-18       MASS_7.3-56            assertthat_0.2.1      
## [121] rjson_0.2.21           withr_2.5.0            sctransform_0.3.5     
## [124] GenomeInfoDbData_1.2.8 parallel_4.2.0         tidyr_1.2.0           
## [127] rmarkdown_2.14         carData_3.0-5          Rtsne_0.16            
## [130] spatstat.explore_3.0-5 shiny_1.7.1            ggbeeswarm_0.6.0