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

Description

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.

Steps to reproduce

For RNA-seq, A total of 1 × 10⁵ P14 cells were sorted and lysed in lysis buffer (100 mM Tris-HCl, pH 7.5; 500 mM LiCl; 10 mM EDTA, pH 8; 1.5% SDS) containing oligo-dT magnetic beads conjugated with Oligo-dT25. The mixture was incubated for five minutes to facilitate mRNA binding. Subsequently, the sample was placed on a magnetic rack to separate the beads, and the supernatant was discarded. The beads were washed twice with Wash Buffer A (10 mM Tris-HCl, pH 7.5; 0.15 M LiCl; 1 mM EDTA, pH 8; 0.1% SDS) and once with Wash Buffer B (10 mM Tris-HCl, pH 7.5; 0.15 M LiCl; 1 mM EDTA, pH 8) to remove non-specific contaminants. The supernatant was carefully removed after each wash. Elution of mRNA was achieved by adding 5 μL of Elution Buffer (10 mM Tris-HCl) and incubating the beads at 80°C for two minutes. The mixture was then subjected to magnetic separation, and the supernatant containing the extracted mRNA was collected. The isolated RNA was reverse transcribed into cDNA using a reaction mixture containing dNTPs, oligodT mix, Maxima H Minus Reverse Transcriptase, and RNase inhibitor . A template switch primer was added for synthesizing the second strand of cDNA, followed by 11 cycles of amplification. The DNA fragments were then fragmented, and the library was constructed according to the manufacturer’s procedure using the TruePrep DNA Library Prep Kit V2 for Illumina. The library was purified and subjected to paired-end sequencing (2 × 150 bp) on an Illumina NovaSeq platform. For ATAC-seq, It was performed using the TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, TD501) with minor modifications. Briefly, 50,000 cells were pelleted at 500 g for 5 minutes and lysed on ice for 10 minutes in pre-chilled lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.1% Tween-20, 0.01% Digitonin and 0.1% IGEPAL CA-630). Nuclei were harvested and subjected to a transposition reaction. The tagmented DNA was purified using the MinElute kit (Qiagen, 28004). Nextera indexing was performed on the tagmented DNA, which was amplified for 11 cycles, using a BIO-GENER GE4851T PCR machine. Amplified products were purified with VAHTS DNA Clean Beads, washed twice with 80% ethanol, and eluted in DEPC-treated Water. Libraries were sequenced on an Illumina NovaSeq platform, generating 150 bp paired-end reads. 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 and aligned to the mouse genome (GRCm38.p6) using Bowtie2 . Duplicate reads were marked using Picard, and only non-duplicated, properly paired reads were retained using samtools . Reads were adjusted for Tn5 transposase shifts, pooled by sample type, and peaks were called using MACS2. Differentially accessible (DA) peaks were generated using dba.analyze function in Diffbind.

Institutions

• Xiamen University

  Fujian, Xiamen

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