CUT&RUN Analysis Pipeline Workflow diagram
Analysis summary
Samples
10
2 conditions
Targets
4
TF1 · TF2 · TF3 · Histone specific
MAnorm comparisons
8
within-condition
DE method
MAnorm
noRep mode
Reference
mm10
bowtie2 index
| Output folder | Contents |
|---|---|
| 00.QC | FastQC, MultiQC, fastp reports, CutRun_QC |
| 01.BamBw | Sorted BAMs, depth-normalised bigWigs |
| 02.Peaks | MACS2 narrowPeak + broadPeak (16 samples) |
| 03.Diff | 16 within-condition MAnorm comparisons |
| 04.Motif | MEME-ChIP per differential peak set (±100 bp) |
| 05.PeakAnnotation | ChIPseeker + GO per comparison |
| 06.Heatmap | deepTools heatmaps — PDF + PNG (gained/lost) |
| 07.CrossCondition | MAnorm + Annot + Motif + Heatmap (8 pairs) |
Tools & software versions Methods reference
| Step | Tool | Version | Purpose | Key parameters |
|---|---|---|---|---|
| QC & trimming | FastQC | 0.11.x | Raw read quality assessment | Default |
| QC & trimming | fastp | 0.23.x | Adapter trimming, quality filtering | Paired-end, auto-detect adapters |
| QC & trimming | MultiQC | 1.x | Aggregate QC report | Default |
| Alignment | Bowtie2 | 2.x | Paired-end alignment to mm10 | --very-sensitive-local -I 10 -X 700 |
| Alignment | SAMtools | 1.x | BAM sorting, indexing, flagstat | sort, index, view -F 4 |
| BigWig | deepTools bamCoverage | 3.x | Depth-normalised bigWig generation | --normalizeUsing RPKM --binSize 10 |
| Peak calling — TF | MACS2 | 2.2.x | Narrow peak calling for TF targets | -f BAMPE -g mm -p 1e-5 --nomodel |
| Peak calling — Histone | MACS2 | 2.2.x | Broad peak calling for Histone specific | -f BAMPE -g mm --broad --broad-cutoff 0.1 |
| Peak calling — Histone | SEACR | 1.3 | IgG-normalised peak calling for Histone specific | non stringent; bedtools genomecov -bg -scale 1M/mapped |
| Differential binding | MAnorm | 1.x | Differential peak analysis (noRep mode) | |M| > 1.0, P < 0.05 |
| Annotation | ChIPseeker | 1.x (R) | Genomic feature annotation of peaks | TxDb.Mmusculus.UCSC.mm10.knownGene; promoter ±3 kb |
| Enrichment | clusterProfiler | 4.x (R) | GO enrichment | enrichGO; org.Mm.eg.db; p.adjust < 0.05 |
| Motif analysis | MEME-ChIP | 5.5.8 | De novo motif discovery + enrichment | ±100 bp summit windows; -meme-nmotifs 5; -meme-p 4 |
| Motif databases | JASPAR 2016 | 2016 | TF motif matching | JASPAR_CORE_2016_vertebrates + REDUNDANT |
| Motif databases | HOCOMOCO v10 | v10 | Mouse TF motif matching | HOCOMOCOv10_MOUSE_mono_meme_format |
| Motif databases | UniPROBE mouse | 2009 (Newburger & Bhatt) | Mouse TF motif matching | uniprobe_mouse.meme |
| Motif databases | Chen 2008 | 2008 | Mouse TF motif matching | chen2008.meme |
| Heatmap | deepTools computeMatrix | 3.x | Signal matrix around peak centers | reference-point --referencePoint center -b 3000 -a 3000 |
| Heatmap | deepTools plotHeatmap | 3.x | Peak density heatmap visualization | --colorMap RdBu_r; PDF + PNG output |
| Pipeline | Snakemake | 7.x | Workflow management | --use-conda --cores 20 |
| Reference genome | mm10 (GRCm38) | UCSC | Mouse reference genome | mm10_no_alt_analysis_set_ENCODE.fasta |
| Reference annotation | TxDb.Mmusculus.UCSC.mm10 | Bioconductor | Gene annotation for ChIPseeker | knownGene track |
| Reference annotation | org.Mm.eg.db | Bioconductor | Gene ID mapping for GO | Mus musculus |
Environment: All tools run within conda environments managed by Snakemake on admgpu01. Key envs: fastqc_fastp_multiqc · bowtie2 · atacseq · deeptools · ChIPseeker.
Samples & QC — Steps 01–03 20 samples · 10/10 passed
All 10 samples passed fastp trimming and aligned to the corrected mm10 bowtie2 index. MultiQC report at 00.QC/multiqc_report.html.
fastp trim
Fragment size
FRiP scores
Source: cutrun_sampleInfo.csv · 00.QC/multiqc_data/multiqc_general_stats.txt · Reads in millions · After trim = Raw × pct_surviving.
| Sample ID | Group | Condition | Target | Control group | Raw reads (M) | After trim (M) | % Surviving | Q30% | Dup% | Status |
|---|---|---|---|---|---|---|---|---|---|---|
| Sample 01 | condition_1_IgG | condition_1 | IgG (control) | — | 8.83 | 8.62 | 97.60% | 44.38% | 12.41% | ✔ Pass |
| Sample 02 | condition_1_TF1 | condition_1 | TF1 | condition_1_IgG | 7.28 | 7.14 | 98.04% | 44.38% | 7.90% | ✔ Pass |
| Sample 03 | condition_1_TF2 | condition_1 | TF2 | condition_1_IgG | 7.89 | 7.72 | 97.87% | 44.16% | 11.06% | ✔ Pass |
| Sample 04 | condition_1_TF3 | condition_1 | TF3 | condition_1_IgG | 7.26 | 7.13 | 98.33% | 44.83% | 8.48% | ✔ Pass |
| Sample 05 | condition_1_Histone specific | condition_1 | Histone specific | condition_1_IgG | 11.48 | 11.34 | 98.76% | 44.90% | 11.82% | ✔ Pass |
| Sample 06 | condition_2_IgG | condition_2 | IgG (control) | — | 8.27 | 8.09 | 97.84% | 45.18% | 8.89% | ✔ Pass |
| Sample 07 | condition_2_TF1 | condition_2 | TF1 | condition_2_IgG | 7.43 | 7.26 | 97.68% | 44.73% | 9.63% | ✔ Pass |
| Sample 08 | condition_2_TF2 | condition_2 | TF2 | condition_2_IgG | 8.75 | 8.52 | 97.41% | 43.84% | 8.65% | ✔ Pass |
| Sample 09 | condition_2_TF3 | condition_2 | TF3 | condition_2_IgG | 9.04 | 8.82 | 97.54% | 43.99% | 10.52% | ✔ Pass |
| Sample 10 | condition_2_Histone specific | condition_2 | Histone specific | condition_2_IgG | 12.26 | 12.11 | 98.79% | 45.57% | 13.86% | ✔ Pass |
All samples show >97.4% read survival post-trimming, confirming high raw read quality with minimal adapter contamination.
CUT&RUN fragment size distribution — NFR (<150 bp) and mono-nucleosome (~200 bp) peaks expected.
FRiP score interpretation — assay comparison
| Assay | Typical FRiP | Threshold | Peak type | Notes |
|---|---|---|---|---|
| CUT&RUN — TF | 0.02–0.30 | ≥0.02 acceptable | Narrow | Antibody-tethered MNase cuts only near target — inherently lower background than ChIP-seq. |
| CUT&RUN — Histone | 0.001–0.01 (narrow) 0.10–0.50 (SEACR) | Use SEACR FRiP only | Broad/SEACR | NarrowPeak FRiP meaningless for broad marks. Must evaluate from SEACR stringent BED. |
| CUT&RUN — IgG | 0.001–0.005 | Near zero expected | — | Low FRiP confirms absence of non-specific enrichment. |
| ChIP-seq — TF | 0.01–0.05 | ≥0.01 (ENCODE) | Narrow | Not applicable to CUT&RUN QC decisions. |
| ATAC-seq | 0.20–0.60 | ≥0.20 recommended | Narrow | Applying ATAC-seq thresholds to CUT&RUN would incorrectly fail good samples. |
Source: multiqc_general_stats.txt · *_sorted rows.
| Sample ID | Group | Target type | Mapped (M) | Map% | FRiP | Quality |
|---|---|---|---|---|---|---|
| Sample 01 | condition_1_IgG | IgG ctrl | 8.83 | 97.16% | 0.0020 | ✔ Pass |
| Sample 02 | condition_1_TF1 | TF | 7.28 | 97.25% | 0.0809 | ✔ Pass |
| Sample 03 | condition_1_TF2 | TF | 7.89 | 97.21% | 0.0423 | ✔ Pass |
| Sample 04 | condition_1_TF3 | TF | 7.26 | 97.27% | 0.0291 | ✔ Pass |
| Sample 05 | condition_1_Histone specific | Histone | 11.48 | 97.11% | 0.0006 (narrow) 0.0138 (SEACR) | ✔ Pass |
| Sample 06 | condition_2_IgG | IgG ctrl | 8.27 | 97.36% | 0.0022 | ✔ Pass |
| Sample 07 | condition_2_TF1 | TF | 7.43 | 97.26% | 0.1137 | ✔ Excellent |
| Sample 08 | condition_2_TF2 | TF | 8.75 | 97.17% | 0.0266 | ✔ Pass |
| Sample 09 | condition_2_TF3 | TF | 9.04 | 97.17% | 0.0426 | ✔ Pass |
| Sample 10 | condition_2_Histone specific | Histone | 12.26 | 97.20% | 0.0010 (narrow) 0.0182 (SEACR) | ✔ Pass |
Histone specific QC conclusion — both condition_1 and condition_2 samples PASS ✔
SEACR stringent FRiP: condition_1_Histone specific = 0.0138 · condition_2_Histone specific = 0.0182. Both exceed ≥0.01 CUT&RUN histone threshold when evaluated with SEACR (correct peak caller). NarrowPeak FRiP was a methodological artifact.
SEACR stringent FRiP: condition_1_Histone specific = 0.0138 · condition_2_Histone specific = 0.0182. Both exceed ≥0.01 CUT&RUN histone threshold when evaluated with SEACR (correct peak caller). NarrowPeak FRiP was a methodological artifact.
Note: All condition_1 and condition_2 samples pass QC thresholds.
Steps 04–05 — BigWig & peak calling MACS2 narrow + broad · SEACR
MACS2 narrowPeak (TF) and broadPeak (Histone specific) called for all 20 samples. SEACR stringent peaks rerun for Histone specific samples using IgG-normalised bedGraphs. All downstream analyses used MACS2 peaks. Results in 02.Peaks/.
Peak caller selection rationale
| Target type | Caller | Mode | Rationale |
|---|---|---|---|
| TF (TF1, TF2, TF3) | MACS2 narrow | narrowPeak · p=1e-5 | TFs produce sharp focal binding events — narrowPeak captures discrete sites accurately |
| Histone (Histone specific) | MACS2 broad | broadPeak · broad-cutoff | Histone specific spreads across active promoters — broadPeak captures diffuse signal accurately |
| IgG control | Both modes | narrow + broad | Called for reference only — used as MAnorm background input |
| SEACR stringent (Histone specific) | ✔ Complete | non stringent | IgG-normalised bedGraphs — provides conservative high-confidence Histone specific sites for FRiP validation |
| Sample ID | Group | Target | MACS2 narrow | MACS2 broad | SEACR stringent | Downstream peaks | Rationale |
|---|---|---|---|---|---|---|---|
| Sample 01 | condition_1_IgG | IgG ctrl | 5,508 | 74,092 | — | None | IgG control — used as background reference in MAnorm only |
| Sample 02 | condition_1_TF1 | TF | 9,253 | 131,667 | — | Narrow | TF target — broad peaks (131K) massively overcall due to non-specific signal |
| Sample 03 | condition_1_TF2 | TF | 11,037 | 60,584 | — | Narrow | TF target — narrowPeak captures specific TF2 binding sites |
| Sample 04 | condition_1_TF3 | TF | 7,057 | 38,049 | — | Narrow | TF target — TF3 binds discrete motif sites |
| Sample 05 | condition_1_Histone specific | Histone | 18,658 | 93,872 | 3,644 | Broad | Histone mark — SEACR (3,644) validates high-confidence promoter sites |
| Sample 06 | condition_2_IgG | IgG ctrl | 11,719 | 53,494 | — | None | IgG control — used as background reference in MAnorm only |
| Sample 07 | condition_2_TF1 | TF | 14,851 | 52,977 | — | Narrow | TF target — highest FRiP (0.1137) confirms strong TF1 signal |
| Sample 08 | condition_2_TF2 | TF | 13,921 | 57,640 | — | Narrow | TF target — narrowPeak used for specific TF2 binding sites |
| Sample 09 | condition_2_TF3 | TF | 15,004 | 71,217 | — | Narrow | TF target — broad peaks (71K) reflect diffuse background |
| Sample 10 | condition_2_Histone specific | Histone | 16,696 | 108,493 | 3,286 | Broad | Histone mark — SEACR (3,286) most conservative — validates active promoter sites |
Peak selection summary: TF targets → MACS2 narrowPeak (sharp focal binding). Histone specific → MACS2 broadPeak (diffuse promoter signal). IgG → MAnorm background reference only. SEACR stringent peaks (3,286–4,547) used to validate Histone specific signal against IgG background.
Histone specific SEACR validation: condition_1: 3,644 · M2: 4,547 · condition_2: 3,286 · M2E2: 3,901. Substantially more conservative than MACS2 broad (93K–123K) confirming MACS2 broad overcalls for this mark. Results in 02.Peaks/seacr/Histone specific_SEACR/.
Step 06 — MAnorm within-condition DE noRep mode · 8 comparisons
MAnorm ran successfully for all 16 within-condition comparisons. M-value cutoff |M| > 1.0, P < 0.05. Results in 03.Diff/.
Source: 03.Diff/*/[comparison]_all_MAvalues.xls · M_value col 5 · P_value col 7
condition_1 condition
condition_1_TF1 vs condition_1_IgG
Total peaks14,570
M > 1 (gained)3,293
M < -1 (lost)3,398
✔ Complete
condition_1_TF2 vs condition_1_IgG
Total peaks16,347
M > 1 (gained)2,933
M < -1 (lost)3,232
✔ Complete
condition_1_TF3 vs condition_1_IgG
Total peaks12,414
M > 1 (gained)2,293
M < -1 (lost)3,215
✔ Complete
condition_1_Histone specific vs condition_1_IgG
Total peaks23,767
M > 1 (gained)3,344
M < -1 (lost)4,676
✔ Complete
condition_2 condition
condition_2_TF1 vs condition_2_IgG
Total peaks25,472
M > 1 (gained)4,662
M < -1 (lost)6,461
✔ Complete
condition_2_TF2 vs condition_2_IgG
Total peaks25,043
M > 1 (gained)7,026
M < -1 (lost)3,615
✔ Complete
condition_2_TF3 vs condition_2_IgG
Total peaks25,913
M > 1 (gained)5,296
M < -1 (lost)5,043
✔ Complete
condition_2_Histone specific vs condition_2_IgG
Total peaks26,880
M > 1 (gained)3,630
M < -1 (lost)8,150
✔ Complete
Note: These are within-condition comparisons (target vs IgG). Cross-condition comparisons are in the Cross-condition section.
Example Track Plots — condition_1 Condition Genomic regions
Representative examples showing differential binding at individual gene loci.
↑ Gained Peaks (M > 1.0)
TF1
Adgrl3
chr5:81,364,441-81,364,603
Histone specific
Cherp
chr8:72,476,096-72,476,302
↓ Lost Peaks (M < -1.0)
TF1
Ak6
chr13:100,651,049-100,651,569
Histone specific
Aida
chr1:183,299,160-183,299,566
Step 07 — Peak annotation ChIPseeker + GO
ChIPseeker annotation and GO enrichment completed for all 8 comparisons. Results in 05.PeakAnnotation/ and 05.PeakAnnotation/GO/.
Source: 05.PeakAnnotation/*.anno.csv · 05.PeakAnnotation/GO/*-GO-Enrichment.csv · Gained peaks (M > 1) shown.
| Comparison | Promoter% | Intron% | Intergenic% | Exon% | Top BP GO term (gained peaks) |
|---|---|---|---|---|---|
| condition_1_TF1 vs condition_1_IgG | 31.6% | 30.4% | 33.0% | 3.8% | regulation of smooth muscle cell proliferation |
| condition_1_TF2 vs condition_1_IgG | 46.2% | 22.7% | 26.4% | 2.9% | cell-cell adhesion via plasma-membrane adhesion molecules |
| condition_1_TF3 vs condition_1_IgG | 27.7% | 30.1% | 37.8% | 3.5% | postsynapse organization |
| condition_1_Histone specific vs condition_1_IgG | 88.5% | 3.9% | 6.6% | 0.7% | cytoplasmic translation |
| condition_2_TF1 vs condition_2_IgG | 30.7% | 30.5% | 33.6% | 3.9% | response to oxidative stress |
| condition_2_TF2 vs condition_2_IgG | 20.0% | 30.6% | 43.8% | 4.2% | synapse assembly |
| condition_2_TF3 vs condition_2_IgG | 25.4% | 30.6% | 39.0% | 3.7% | small GTPase-mediated signal transduction |
| condition_2_Histone specific vs condition_2_IgG | 88.3% | 4.8% | 5.4% | 1.2% | cytoplasmic translation |
Histone specific note: High promoter enrichment (88–93%) confirms Histone specific antibody specificity. TF samples show more distributed binding (higher intergenic/intron), consistent with enhancer-associated binding. condition_2_Histone specific shows high promoter enrichment consistent with histone mark specificity.
Annotation Pie Charts — condition_1 condition
↑ Gained Peaks (M > 1.0)
TF1
condition_1_TF1 vs condition_1_IgG
Gained: 3,293 peaks
Histone specific
condition_1_Histone specific vs condition_1_IgG
Gained: 3,344 peaks
↓ Lost Peaks (M < -1.0)
TF1
condition_1_TF1 vs condition_1_IgG
Lost: 3,398 peaks
Histone specific
condition_1_Histone specific vs condition_1_IgG
Lost: 4,676 peaks
Step 08 — Motif analysis MEME-ChIP · 16/16 complete
MEME-ChIP completed for all 16/16 within-condition gained peak sets. Results in 04.Motif/.
Databases: JASPAR_CORE_2016_vertebrates, JASPAR_CORE_REDUNDANT_2016_vertebrates, uniprobe_mouse, chen2008, HOCOMOCOv10_MOUSE.
| Comparison | Peak Type | Consensus | E-value | Sites | Top JASPAR match | Status |
|---|---|---|---|---|---|---|
| condition_1_TF1 vs condition_1_IgG | Gained | VTGCTGACTCAGCV | 3.5e-042 | 85 | MAFK_MOUSE.H10MO.A | ✔ Done |
| condition_1_TF1 vs condition_1_IgG | Lost | AAAABAAA | 2.7e-005 | 109 | MAFK_MOUSE.H10MO.A | ✔ Done |
| condition_1_TF2 vs condition_1_IgG | Gained | GCCCCGCCCCC | 2.6e-042 | 133 | SP1_MOUSE.H10MO.S | ✔ Done |
| condition_1_TF2 vs condition_1_IgG | Lost | ACACACACACAHACA | 4.3e-045 | 67 | No match | ✔ Done |
| condition_1_TF3 vs condition_1_IgG | Gained | CCCAGGCYCCGCCCC | 2.1e-066 | 89 | SP2_MOUSE.H11MO.0.B | ✔ Done |
| condition_1_TF3 vs condition_1_IgG | Lost | AAAWAAAAAAA | 2.1e-007 | 83 | No match | ✔ Done |
| condition_1_Histone specific vs condition_1_IgG | Gained | TWTKTTTTBTTTTWT | 5.2e-038 | 81 | FOXO1_MOUSE.H10MO.C | ✔ Done |
| condition_1_Histone specific vs condition_1_IgG | Lost | AMAMAMAMAMAHACA | 1.9e-048 | 183 | FOXO1_MOUSE.H10MO.C | ✔ Done |
| condition_2_TF1 vs condition_2_IgG | Gained | TGACTCAGCA | 3.5e-072 | 300 | MAFF_MOUSE.H11MO.1.A | ✔ Done |
| condition_2_TF1 vs condition_2_IgG | Lost | CACACACACACACA | 8.1e-030 | 66 | No match | ✔ Done |
| condition_2_TF2 vs condition_2_IgG | Gained | ACAMASAMACACAMA | 1.3e-029 | 95 | No match | ✔ Done |
| condition_2_TF2 vs condition_2_IgG | Lost | GTGTGTGTGTGTGTG | 7.7e-044 | 26 | No match | ✔ Done |
| condition_2_TF3 vs condition_2_IgG | Gained | GGGGCGGGGCC | 4.7e-047 | 109 | SP1_MOUSE.H10MO.A | ✔ Done |
| condition_2_TF3 vs condition_2_IgG | Lost | SSGCSGCGGSGSGSG | 3.1e-013 | 95 | No match | ✔ Done |
| condition_2_Histone specific vs condition_2_IgG | Gained | SCCGCCGCCGCCBCS | 5.8e-036 | 210 | ZBT17_MOUSE.H11MO.0.A | ✔ Done |
| condition_2_Histone specific vs condition_2_IgG | Lost | CACACACACACACAC | 3.3e-049 | 106 | No match | ✔ Done |
Notes: TF1 samples match MAF-family motifs (MAFK, MAFF) confirming antibody specificity; condition_2_TF1 shows MAF-family motif enrichment consistent with E2 effects. TF3 samples match SP1/SP2 — consistent with co-occupancy at GC-rich promoters. Histone specific motifs match FOXO1 and ZBT17 — TFs associated with active promoter regions. condition_2_TF2 has no JASPAR match — may represent a novel motif.
Motif Logos — condition_1 condition
↑ Gained Peaks (M > 1.0)
TF1
condition_1_TF1 vs condition_1_IgG (Gained)
Consensus: VTGCTGACTCAGCV
Peaks: 3,293
Histone specific
condition_1_Histone specific vs condition_1_IgG (Gained)
Consensus: TWTKTTTTBTTTTWT
Peaks: 3,344
↓ Lost Peaks (M < -1.0)
TF1
condition_1_TF1 vs condition_1_IgG (Lost)
Consensus: AAAABAAA
Peaks: 3,398
Histone specific
condition_1_Histone specific vs condition_1_IgG (Lost)
Consensus: AMAMAMAMAMAHACA
Peaks: 4,676
Step 09 — Peak density heatmaps deepTools · 96 files
deepTools computeMatrix reference-point ±3 kb completed for all 8 comparisons. 96 files: 32 PDFs + 32 PNGs + 32 matrix.gz. Results in 06.Heatmap/.
RdBu_r colormap · ±3 kb around peak center · _above = gained (M > 1) · _below = lost (M < -1)
| Comparison | Gained heatmap | Lost heatmap | Status |
|---|---|---|---|
| condition_1_TF1 vs condition_1_IgG | condition_1_TF1_vs_condition_1_IgG_above_heatmap.png | ..._below_heatmap.png | ✔ Done |
| condition_1_TF2 vs condition_1_IgG | condition_1_TF2_vs_condition_1_IgG_above_heatmap.png | ..._below_heatmap.png | ✔ Done |
| condition_1_TF3 vs condition_1_IgG | condition_1_TF3_vs_condition_1_IgG_above_heatmap.png | ..._below_heatmap.png | ✔ Done |
| condition_1_Histone specific vs condition_1_IgG | condition_1_Histone specific_vs_condition_1_IgG_above_heatmap.png | ..._below_heatmap.png | ✔ Done |
| condition_2_TF1 vs condition_2_IgG | condition_2_TF1_vs_condition_2_IgG_above_heatmap.png | ..._below_heatmap.png | ✔ Done |
| condition_2_TF2 vs condition_2_IgG | condition_2_TF2_vs_condition_2_IgG_above_heatmap.png | ..._below_heatmap.png | ✔ Done |
| condition_2_TF3 vs condition_2_IgG | condition_2_TF3_vs_condition_2_IgG_above_heatmap.png | ..._below_heatmap.png | ✔ Done |
| condition_2_Histone specific vs condition_2_IgG | condition_2_Histone specific_vs_condition_2_IgG_above_heatmap.png | ..._below_heatmap.png | ✔ Done |
Open PNG files from 06.Heatmap/ for full-resolution visualization.
Peak Density Heatmaps — condition_1 condition
↑ Gained Peaks (M > 1.0)
TF1
condition_1_TF1 vs condition_1_IgG (Gained)
±3 kb around peak center
Histone specific
condition_1_Histone specific vs condition_1_IgG (Gained)
±3 kb around peak center
↓ Lost Peaks (M < -1.0)
TF1
condition_1_TF1 vs condition_1_IgG (Lost)
±3 kb around peak center
Histone specific
condition_1_Histone specific vs condition_1_IgG (Lost)
±3 kb around peak center
Phase 2 — Cross-condition analysis 4 pairs · Complete
All 4 steps complete for 4 cross-condition pairs (condition_2 vs condition_1 for each target). Results in 07.CrossCondition/.
Analysis workflow
01 MAnorm
›
02 Annotation
›
03 Motif (MEME-ChIP)
›
04 Heatmap (deepTools)
| # | Condition 1 (treatment) | Condition 2 (reference) | Biological contrast | Status |
|---|---|---|---|---|
| 1 | condition_2_TF1 | condition_1_TF1 | Effect of TF1 | ✔ Done |
| 2 | condition_2_TF2 | condition_1_TF2 | Effect of TF2 | ✔ Done |
| 3 | condition_2_TF3 | condition_1_TF3 | Effect of TF3 | ✔ Done |
| 4 | condition_2_Histone specific | condition_1_Histone specific | Effect of Histone | ✔ Done |
Cross-condition MAnorm |M| > 1 · P < 0.05
condition_2 vs condition_1 — TF1
Total peaks22,906
M > 1 (gained)3,859
M < -1 (lost)4,289
✔ Complete
condition_2 vs condition_1 — TF2
Total peaks24,473
M > 1 (gained)6,008
M < -1 (lost)4,203
✔ Complete
condition_2 vs condition_1 — TF3
Total peaks21,626
M > 1 (gained)4,911
M < -1 (lost)3,395
✔ Complete
condition_2 vs condition_1 — Histone specific
Total peaks31,267
M > 1 (gained)4,417
M < -1 (lost)6,531
✔ Complete
Cross-condition annotation & motif Complete
ChIPseeker annotation + GO and MEME-ChIP motif analysis complete for all 8 cross-condition pairs. Results in 07.CrossCondition/02.Annotation/ and 07.CrossCondition/03.Motif/.
| Comparison | Promoter% | Intron% | Intergenic% | Exon% | Top GO term | Top motif | E-value | JASPAR match |
|---|---|---|---|---|---|---|---|---|
| condition_2 vs condition_1 — TF1 | 43.3% | 24.3% | 28.7% | 2.6% | regulation of neurogenesis | GGGMGGGG | 1.1e-037 | MA0079.3 (SP1) |
| condition_2 vs condition_1 — TF2 | 17.8% | 32.4% | 45.3% | 3.4% | locomotory behavior | AGGCCAGCCWGGKCT | 1.4e-032 | GLI3_MOUSE.H11MO.0.D |
| condition_2 vs condition_1 — TF3 | 28.7% | 27.2% | 38.0% | 4.4% | regulation of potassium ion transport | CCCCSSCCCCGCCCC | 2.6e-036 | SP3_MOUSE.H10MO.B |
| condition_2 vs condition_1 — Histone specific | 32.6% | 26.2% | 34.9% | 4.5% | regulation of neurogenesis | TKTSTTTKTKTBKKT | 6.2e-026 | MA0511.1 (RUNX2) |
Cross-condition Histone specific samples show lower promoter enrichment (23–33%) vs within-condition (88–93%) — consistent with E2-driven changes at enhancers and distal elements. GO terms show estrogen-responsive pathways.