Summary of Processed Multi-Omics and GSEA Results in KLF2-Deficient and KLF2-Overexpressing Cells

For RNA-seq analyses, sequencing reads were aligned to the GRCm38.p6 mouse reference genome using STAR. Gene-level read counts (raw counts) were summarized using featureCounts. Differential expression analysis was conducted with DESeq2, which uses a negative binomial distribution for statistical modeling. The Benjamini-Hochberg method was applied to adjust p-values for multiple comparisons. Gene set enrichment analysis (GSEA) and functional gene set enrichment were conducted using the gseapy, utilizing the C7 Immunological Signatures collections from the Molecular Signatures Database.
For ATAC-seq, Activated P14 cells transduced with empty vector- or Klf2-expressing vector were cultured and expanded in vitro for 2.5 days. cells were sorted for ATAC-seq. In brief, 2×150-bp paired-end reads obtained from NovaSeq were trimmed for Nextera adaptors using cutadapt (v4.8, with parameters -m 36 -n 3 -q 10) and aligned to the mouse genome (GRCm38.p6) using Bowtie2 (v2.5.1, with parameters --very-sensitive -X 2000 --no-mixed --no-discordant). Duplicate reads were marked using Picard (v2.9.5, https://broadinstitute.github.io/picard/), and only non-duplicated, properly paired reads were retained using samtools (v1.18, with parameters --F 1804 -f 2 --q 20). Reads were adjusted for Tn5 transposase shifts (+4 bp for the sense strand and −5 bp for the antisense strand), pooled by sample type, and peaks were called using MACS2 (v2.2.7.1, with parameters -q 0.05 --nolambda --keep-dup all --call-summits -f BAMPE). Peaks were finalized by retaining those with higher cut-offs (MACS2 -q 0.05) and were subsequently consolidated into reproducible consensus peaks across replicates (≥50% reproducibility) using Diffbind (v2.10.0, https://bioconductor.org/packages/release/bioc/html/DiffBind.html ). Differentially accessible (DA) peaks were generated using dba.analyze function (bFullLibrarySize = FALSE ) in Diffbind.
Gene set enrichment analysis (GSEA) and functional gene set enrichment were conducted using the gseapy, utilizing the C7 Immunological Signatures collections from the Molecular Signatures Database.