About this repository

This repository contains the source codes for the sequence analysis used in the following paper. Enhanced transcriptional heterogeneity mediated by NF-κB super-enhancers (Wibisana et al., 2022)

PLoS Genetics link

Relative paths under src are shown for codes and under data for data files.

scRNA-seq analysis (1_RNAseq)

Preprocessing data and alignment

• 1-1_preprocessing_alignment
  • 1_trimming_qc_alignment.sh

Downstream analysis (Clustering, pseudodose analysis, etc.)

• 1-2_singlecell_analysis
  • 1_scrnaseq_analysis.R
  • 2_pseudotime.R
  • 3_Fano_factor_calculations.R
  • 4_hillanalysis.R
  • 5_motif_counting_analysis.R
  • mito_genes.R (Function to retrieve mitochondrial gene annotations)

scATAC-seq analysis (2_ATACseq)

Preprocessing of GTF file, FastQ data sampling and cellranger-atac pipeline

• 2-1_preprocessing_alignment_pipeline
  • 1_preprocessing_pipeline.sh
  • ggallus.config (cellranger-atac configuration file)
• 2-2_peak_calling_manipulation
  • 1_peak_calling.sh
  • 2_merge_bed_annotate.sh
• 2-3 SE_analysis
  • 1_SE_analysis.R

Co-accessibility analysis

• 2-4_coaccessibility analysis
  • 0_1_chr_length.sh (get chromosome length from fasta file)
  • 1_assign_fragments.R
  • 2_cicero_main.R
  • 3_cicero_analyze_change.R
  • 4_annotate_conns.sh

Motif analysis

• 2-5_motif_analysis
  • 1_homer_to_fimo.R (Convert Homer motif files to Fimo motif files)
  • 2_FIMO_motif_analysis.sh
  • 3_homer_motif_analysis.sh

Primary B cell analysis

• 2-6_primary_b_cell
  • 1_download_files.sh
  • 2_mapping_atac.sh

scATAC-seq and scRNA-seq combined analysis (3_RNA_atac)

• 1_GO_analysis.R
• 2_Fano_analysis.R

Foci fitting (4_foci)

• 1_logistic_regression.R
• 2_hill_fitting.R

Data files

RelA-GFP foci data (1_foci)

• 1_foci_quant.csv (All quantified foci across all dose and time points)
• 2_foci_20min.csv (Quantified foci at 20 minutes across all dose points)
• 3_median_fitting.csv (Median quantified foci at 20 minutes across all dose points for fitting)

RNA-FISH data (2_RNA_FISH)

• CD83.csv
• NFKBIA.csv

For both files, the first column dose contains information of the anti-IgM concentration. Column CELL contains unique cell identifier per viewpoint. N_Q570 is the target RNA spot number, while N_Q670 is GAPDH. Column GFP contains RelA-GFP foci number measured using the same method with RNA-FISH spot quantification.