OVERVIEW The following README contains: i) the instruction to set up an R environment (R version 3.6.3) and to install R Studio on a Windows/macOS operative system; ii) the instructions to install Seurat 3.1.5 along with all its dependencies; iii) the software code used to generate all the plots and figure as showed in the manuscript. SYSTEM REQUIREMENTS The following analysis can be performed on a standard computer with sufficient RAM to support the in-memory operation required by Seurat 3.1.5. The installation of Seurat 3.1.5 and all its dependencies have been tested on both Windows (version 10 64-bit ) and macOS (Big Sur version 11.6.1) operating systems. ALL PACKAGES and DEPENDENCIES attached base packages: [1] stats graphics grDevices utils datasets methods base other attached packages: [1] biomaRt_2.42.1 Seurat_3.1.5 Matrix_1.4-0 [1] TH.data_1.1-0 Rtsne_0.15 colorspace_2.0-2 ellipsis_0.3.2 ggridges_0.5.3 leiden_0.3.9 [7] listenv_0.8.0 remotes_2.4.2 ggrepel_0.9.1 bit64_4.0.5 AnnotationDbi_1.48.0 fansi_1.0.2 [13] mvtnorm_1.1-3 mathjaxr_1.4-0 codetools_0.2-18 splines_3.6.3 mnormt_2.0.2 cachem_1.0.6 [19] TFisher_0.2.0 jsonlite_1.7.3 ica_1.0-2 dbplyr_2.1.1 cluster_2.1.2 png_0.1-7 [25] uwot_0.1.11 sctransform_0.3.3 BiocManager_1.30.16 compiler_3.6.3 httr_1.4.2 assertthat_0.2.1 [31] fastmap_1.1.0 lazyeval_0.2.2 cli_3.1.1 prettyunits_1.1.1 htmltools_0.5.2 tools_3.6.3 [37] rsvd_1.0.5 igraph_1.2.11 gtable_0.3.0 glue_1.6.1 RANN_2.6.1 reshape2_1.4.4 [43] dplyr_1.0.7 rappdirs_0.3.3 Rcpp_1.0.8 Biobase_2.46.0 vctrs_0.3.8 multtest_2.42.0 [49] ape_5.6-1 nlme_3.1-155 lmtest_0.9-39 stringr_1.4.0 globals_0.14.0 rbibutils_2.2.7 [55] lifecycle_1.0.1 irlba_2.3.5 XML_3.99-0.3 future_1.23.0 MASS_7.3-55 zoo_1.8-9 [61] scales_1.1.1 hms_1.1.1 parallel_3.6.3 sandwich_3.0-1 RColorBrewer_1.1-2 curl_4.3.2 [67] memoise_2.0.1 reticulate_1.24 pbapply_1.5-0 gridExtra_2.3 ggplot2_3.3.5 stringi_1.7.6 [73] RSQLite_2.2.7 mutoss_0.1-12 S4Vectors_0.24.4 plotrix_3.8-2 BiocGenerics_0.32.0 Rdpack_2.1.3 [79] rlang_1.0.1 pkgconfig_2.0.3 matrixStats_0.61.0 lattice_0.20-45 ROCR_1.0-11 purrr_0.3.4 [85] patchwork_1.1.1 htmlwidgets_1.5.4 cowplot_1.1.1 bit_4.0.4 tidyselect_1.1.1 parallelly_1.30.0 [91] RcppAnnoy_0.0.19 plyr_1.8.6 magrittr_2.0.2 R6_2.5.1 IRanges_2.20.2 generics_0.1.2 [97] multcomp_1.4-18 DBI_1.1.2 pillar_1.7.0 sn_2.0.1 fitdistrplus_1.1-6 survival_3.2-13 [103] tibble_3.1.6 future.apply_1.8.1 tsne_0.1-3 crayon_1.4.2 KernSmooth_2.23-20 utf8_1.2.2 [109] BiocFileCache_1.10.2 tmvnsim_1.0-2 plotly_4.10.0 progress_1.2.2 grid_3.6.3 data.table_1.14.2 [115] blob_1.2.2 metap_1.7 digest_0.6.29 tidyr_1.2.0 numDeriv_2016.8-1.1 openssl_1.4.6 [121] stats4_3.6.3 munsell_0.5.0 viridisLite_0.4.0 askpass_1.1 SOFTWARE INSTALLATION GUIDE 1. R version 3.6.3 for Windows and for macOS can be downloaded at: https://cran.r-project.org/bin/windows/base/old/3.6.3Test/ (Windows) and https://cran.r-project.org/bin/macosxTest/ (macOS). 2. Packages “remotes”, “BiocManager”, “biomaRt”, “Matrix” can be installed running the command: install.packages() from R console. 3. Seurat3.1.5 and all its dependencies can be installed running the following command line: remotes::install_version("Seurat", version = "3.1.5") 4. A typical installation time on a “normal” desktop computer is around 15-20 minutes. INSTRUCTION FOR USE/EXPECTED OUTPUT/EXPECTED RUN TIME All the instructions to install and run the code on the data provided in this folder are in the document “script_NC.txt”. The expected outputs of the code are: 1) Supplementary tables 2-7; 2) Figure 5 (A-D); 3) Supplementary figures 9-11. Expected run time is about 45 min for all the analyses.
