This code supplements the publication by Fetahu, Esser-Skala, Dnyansagar et al (2023).

(Not all of these folders appear in the git repository.)

• data_generated: output files generated by the scripts in this repository
• data_raw: raw input data
• doc: project documentation
• literature: relevant publications
• metadata: additional required data
• misc: miscellaneous scripts
• plots: generated plots
• renv: R environment data
• scatac: scripts for scATAC-seq analysis
• tables: exported supplementary tables and data; the subfolder source_data contains source data for figures

Create a folder data_raw that will contain raw data in the following subfolders:

• adrmed:
  • adrenal_medulla_Seurat.RDS: reference expression data for adrenal medullary cells; download from https://adrenal.kitz-heidelberg.de/developmental_programs_NB_viz/ (Download data -> Download Adrenal medulla data -> Seurat object (RDS))
• rna_seq: Download GSE216155_RAW.tar from GEO Series GSE216155 and extract all files.
• atac_seq: Download all files from GEO Series GSE216175 (GSE216175_barcodes.tsv.gz, GSE216175_barcodes_samples.csv.gz, GSE216175_filtered_peak_bc_matrix.h5, GSE216175_matrix.mtx.gz, GSE216175_peaks.bed.gz, and GSE216175_RAW.tar). Extract all files from the tarball.
• GSE137804: download the following files from GEO series GSE137804:
  • GSE137804_tumor_dataset_annotation.csv.gz
  • GSE137804_RAW.tar, from which the following eleven files must be extracted:
    • GSM4088774_T10_gene_cell_exprs_table.xls.gz
    • GSM4088776_T27_gene_cell_exprs_table.xls.gz
    • GSM4088777_T34_gene_cell_exprs_table.xls.gz
    • GSM4088780_T69_gene_cell_exprs_table.xls.gz
    • GSM4088781_T71_gene_cell_exprs_table.xls.gz
    • GSM4088782_T75_gene_cell_exprs_table.xls.gz
    • GSM4088783_T92_gene_cell_exprs_table.xls.gz
    • GSM4654669_T162_gene_cell_exprs_table.xls.gz
    • GSM4654672_T200_gene_cell_exprs_table.xls.gz
    • GSM4654673_T214_gene_cell_exprs_table.xls.gz
    • GSM4654674_T230_gene_cell_exprs_table.xls.gz
• snp_array: Extract the contents of snp_array.tgz provided in Zenodo repository https://doi.org/10.5281/zenodo.7707614

Optionally, obtain intermediary data: Extract the contents of R_data_generated.tgz from Zenodo repository https://doi.org/10.5281/zenodo.7707614 to folder data_generated.

Run these R scripts in the given order to generate all files required by figures and tables.

• perform_qc.R – perform QC filtering, ensure unique cell names
• integrate_rna.R – integrate scRNA-seq samples with monocle, perform basic analyses and extract resulting metadata
• correct_ambiance.R – remove cell-free RNA contamination
• classify_cell_types.R – perform cell type classification via SingleR
• assemble_metadata.R – generate one CSV and RDS file with all metadata
• analyse_dge.R – analyse differential gene expression using mixed models
• analyse_cnv.R – analyse copy number variations
• analyse_ccc.R – analyse cell-cell communication
• analyse_myeloid.R – analyse myeloid subpopulation
• prepare_data_dong.R – prepare dataset by Dong et al for analysis
• classify_as_adrenal.R – classify tumor cells as adrenal medullary cell types
• compare_tumors_pseudobulk.R – comparison of tumor samples via pseudobulk correlation

Run these R scripts in arbitrary order to generate publication figures and tables:

• plot_figure_1_S1.R – includes Tables S2, S4 and Data S1
• plot_figure_2_S2.R
• plot_figure_3_S3.R – includes Data S2
• plot_figure_4_S4_S7b.R – includes Data S3 and S4
• plot_figure_S5_S7c.R – includes Data S5 and S6
• plot_figures_revision.R – plots for the reply to reviewers

• common_functions.R – functions used throughout the project
• plot_dependencies.R – plot the dependency graph
• styling.R – functions for generating publication-quality figures and tables

All required scripts are in subfolder scatac.

• R0_scopen.R – quality control, normalization, clustering
• R1_annotation.R – annotation of clusters formed and markers used
• R2_foot_printing_cell_type.R – cell-type specific footprinting
• R3_nebula_run.R – identify differentially accessible OCRs in patient groups
• R4_nebula_results.R – analyze nebula results
• R5_nebula_microenvironment.R – enrichment in microenvironment cells
• R6_nebula_nbcells.R – enrichment in NB cells
• R7_create_bed_run_homer.R – create cell specific bed files and motif analysis
• R8_homer_results.R – analyze motif analysis results

For data integration we used scGLUE (Graph Linked Unified Embedding) model for unpaired single-cell multi-omics data integration (https://scglue.readthedocs.io/en/latest/). We followed the detailed tutorial at https://scglue.readthedocs.io/en/latest/tutorials.html. Before the tutorial we needed to convert the objects in anndata format from SingleCellExperiment and Seurat for scRNA-seq and scATAC-seq respectively. There are many tools available to do this and we are sharing our approach for format conversion, namely monocle_to_anndata.R and Seurat_to_anndata.R.

The following Jupyter notebooks follow the notebooks of the scGLUE integration pipeline.

• G1_nb_glue_preprocessing_myeloid.ipynb – preprocess scRNA-seq and scATAC-seq anndata objects
• G2_glue_model_myeloid.ipynb – create glue model
• G3_regulatory_inference_myeloid.ipynb
• G4_regulatory_network_plots_myeloid.ipynb

Finally, Figures.R generates publication figures.