This repository contains analysis codes used for the publication: Sex-differences-orchestrated-by-androgens-at-single-cell-resolution

Codes provided here are to define the DEGs, AASB-DEGs and significantly enriched biological pathways of each cell type across the 17 tissues.

1. Cellranger_pipeline: The code script in Shell for cellranger pipeline which is executed on Linux system.
2. Data_processing: The code in R to perform QC and construct seurat object for downstream analysis.
3. DEG_definition: The code in R to define the DEGs between two different conditions (p-adjust < 0.05 and |Log2FC| > 0.5).
4. AASB-DEG_definition: The code in R to define androgen-associated sex-biased DEGs (AASB-DEGs) for each cell type across the 17 tissues, which comprises positive AASB-DEGs (the expression levels of which are male-biased and positively associated with androgens) and negative AASB-DEGs (the expression levels of which are female-biased and negatively associated with androgens).
5. Pathway_enrichment: The code in R to definine the significantly enriched biological pathways based on DEGs and AASB-DEGs respectively (p < 0.01 & q < 0.01).

Downloading the data

• The raw data for single-cell RNA-seq has been deposited in GSA (https://ngdc.cncb.ac.cn/gsa/) under CRA006610.
• The processed data for single-cell RNA-seq has been deposited in OMIX (https://ngdc.cncb.ac.cn/omix/releaseList) under OMIX001083.

Operation systems

• Linux version 3.10.0-957.el7.x86_64
• Windows 10 64-bit

Hardware requirements

The pipeline requires only a standard computer with enough RAM to support the operations. For optimal performance, we recommended and used a computer with the following specs for testing: AMD Ryzen Threadripper 2950X 16-Core Processor 3.50 GHz and RAM 128 GB.

Installation guide

R packages required for the pipeline can be installed from CRAN (https://cran.r-project.org/) using the install.packages() function, or from Bioconductor (https://bioconductor.org/) using the BiocManager::install() function. The three packages in “05. Pathway_enrichment” should take approximately 11 minutes on a recommended computer for installing.

Packages

• Cell Ranger (version 6.0.2)
• Seurat (version 4.0.3)
• DropletUtils (version 1.18.1)
• DoubletFinder (version 2.0.3)
• ggsci (version 2.9)
• RColorBrewer (version 1.1-3)
• ggplot2 (version 3.4.2)
• dplyr (version 1.0.8)
• BiocParallel (version 1.28.3)
• clustertree (version 0.5.0)
• future (version 1.33.0)
• clusterProfiler (version 3.12.0)
• org.Mm.eg.db (version 3.8.2)
• biomaRt (version 2.40.5)

Demo

1. AASB-DEG_definition: The pipeline to define AASB-DEGs

• Input: required input data files
• Output: expected output results
• The pipeline should take approximately 7 seconds on a recommended computer

2. Pathway_enrichment: The pipeline to perform pathway enrichment analysis of DEGs

• Input: required input data files
• Output: expected output results
• The pipeline should take approximately 4 hours to finish all the analyses on a recommended computer

3. Users can also directly provide their own input files with the format required, and perform analysis by modifying our codes