KOLF2.1J iTF-Microglia: A standardized platform to study microglial transcriptional regulatory networks in CNS disease

All code generated for Rodriguez-Nunez et al. 2025

Link to pre-print: https://www.biorxiv.org/content/10.1101/2025.05.30.657077v1

ABSTRACT

Understanding transcriptional regulatory networks (TRNs) in microglia is key to uncovering mechanisms driving central nervous system (CNS) disorders. Human iPSC-derived models offer a tractable system for studying microglia, yet variability between lines has limited reproducibility. Here, we use the standardized KOLF2.1J iTF line to rapidly generate microglia-like cells (iTF-Microglia) and profile TRNs under homeostatic and inflammatory conditions. iTF-Microglia closely resemble primary brain microglia at both transcriptomic and epigenomic levels. Integrative analyses reveal microglia-enriched candidate cis-regulatory elements (cCREs) and dynamic enhancer remodeling upon differentiation and LPS+IFNG stimulation, involving key transcription factors (TFs) including NF-κB, IRF, and STAT families. TRNs active in iTF-Microglia are enriched for genetic variants linked to Alzheimer’s disease and other CNS disorders. These findings establish KOLF2.1J iTF-Microglia as a reproducible and genetically tractable platform for studying human microglial gene regulation and provide mechanistic insight into how TRN remodeling may contribute to CNS disease risk.

DIRECTORY

Script	Description
20240829_ABC_model_test_KOLF2.1J-iTF.txt	Instructions and job submission scripts for running the ABC Enhancer–Gene Prediction pipeline on KOLF2.1J-iTF microglia data, including conda setup, Snakemake execution, ATAC-seq prep, and Hi-C integration.
ChIPseq_H3K27ac_batch2.txt	Processing notes for the second batch of H3K27ac ChIP-seq including FastQC QC, adapter trimming with BBMap, and downstream Cromwell/WDL runs.
DESeq2_tss_annotation_for_DegCre.R	Prepares DESeq2 differential expression results for DEG–CRE association analysis.
DegCre_analysis_20250427.R	Runs DegCre analysis combining differential expression and chromatin accessibility data; outputs DEG–CRE association plots and statistics.
KOLF2.1J-iTF_paper_ATACseq.txt	Documentation of ATAC-seq processing for iTF iPSCs and microglia . Includes sequencing metadata, QC, Cromwell pipeline runs, and LDSC enrichment.
KOLF2.1J-iTF_paper_ChIPseq_H3K27ac.txt	Metadata and pipeline notes for H3K27ac ChIP-seq experiments in iTF iPSCs and microglia, with replicate details and processing steps.
LDSC_bed_files_20250429.R	Generates LDSC annotation BED files from ATAC-seq/ChIP-seq peaks for partitioned heritability analysis.
LDSC_heatmap.R	Partitioned heritability results and produces ComplexHeatmap-based visualizations of LDSC enrichments across GWAS traits and functional categories .
MAGMA_expression_matrix_format.R	Formats TPM expression matrices for MAGMA input, including averaging replicates, winsorization, and log2 transforms.
MAGMA_plot.R	Reads MAGMA gene-set results and generates publication-quality plots with multiple correction methods applied to GWAS enrichments.
Partitioned_heritability_files.R	Processes LDSC results into partitioned heritability summary files with p-value corrections across GWAS datasets.
RNAseq_analysis.R	Pipeline for bulk RNA-seq analysis of iTF microglia, including DESeq2 differential expression, QC, and visualization.
RNAseq_meta-analyisis_pipeline.txt	Pipeline for meta-analysis of RNA-seq datasets (Dräger, Abud, etc.), with download commands, Salmon/QuantSeq processing, and integration steps.
RNAseq_quantification_pipeline.txt	Job scripts for RNA-seq quantification using STAR, Salmon, and HTSeq; includes references and QC steps.
TF_monaLisa_analysis.R	Uses monaLisa and JASPAR motifs to analyze enriched TF binding motifs in differential peaks; integrates ATAC/ChIP and csaw results.
cCRE-TargetGene_analysis.R	Links candidate cCREs to putative target genes using proximity, RNA-seq, and Hi-C data.
cCRE_analysis.R	Identifies and characterizes cis-regulatory elements from ATAC-seq and ChIP-seq, performing overlap, enrichment, and visualization analyses.
cCRE_disease_variant_annotation.R	Annotates cCREs with disease-associated variants (AD, ALS, FTD, etc.), overlaps with ENCODE TF binding, and integrates variant resources.
monalisa_b.R	Secondary monaLisa motif enrichment analysis script, complementary to TF_monaLisa_analysis.
motifbreakR_script.R	Applies motifbreakR to disease SNPs overlapping cCREs to test for TF motif disruptions; outputs per-SNP motif effect predictions.