Experiment / E4WH5N80XEpisomal Plasmid MPRA

CHEQ-seq captured enhancer reporter assay in MCF7

Multiplex enhancer-reporter assays uncover unsophisticated TP53 enhancer logic

Captured genomic fragments centered on TP53 ChIP-seq peaks, together with housekeeping and negative controls, were cloned upstream of a Venus reporter with 17-bp random barcodes and assayed in MCF7 cells under p53-high (wild-type TP53 plus Nutlin-3a) and p53-low (stable TP53 knockdown) conditions. The table contains fragment-level aggregates of uniquely mapped barcodes and matched plasmid/input and cDNA counts.

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Organism: Human
Taxonomy ID: NCBITaxon:9606
Biosample: CVCL:0031
Reference genome: hg19
Design focus: Region-focused
Region of interest: Not reported / not applicable

Perturbation & assay details

p53-high: 5 uM Nutlin-3a for 24 h in MCF7 TP53-wild-type cells; p53-low: stable TP53 knockdown MCF7 cells, untreated

CHEQ-seq used sheared genomic DNA captured with custom MYbaits around the top TP53 ChIP-seq peaks, 94 housekeeping promoters, and 66 negative control regions. Captured fragments (approximately 500-1000 bp sheared input; inserts averaging about 500 bp) were cloned upstream of a minimal promoter, synthetic intron, and Venus reporter with a 17-bp random barcode. PacBio long reads linked barcodes to genomic fragments, and Illumina cDNA barcode counts measured reporter output. Two biological replicates were available for each p53-high and p53-low condition; untreated wild-type plasmid libraries and a common input library are retained as DNA controls.

Processed data

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Visible columns (33 of 33)

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Filters apply to this table only. The CSV download contains the complete processed table; filtered rows are available through the API.

Column dictionary · 33 definitions

fragment_id: Unique packaged fragment identifier.
feature_name: Original named target when present, such as a MACS peak or control; blank for an unlabeled genomic fragment.
coordinate: hg19 coordinate in chr:start-end form for an interval or chr:start form for a named point.
coordinate_type: Whether the record is a mapped genomic fragment or a named target point.
chrom: Chromosome or contig from the PacBio genomic mapping.
start: Zero-based genomic start coordinate from the mapped fragment or named point.
end: Genomic end coordinate for an interval; blank for a named point.
n_barcodes: Number of uniquely mapped barcodes aggregated for the fragment locus after cDNA QC.
input_count_sum: Sum of supplied counts across the common input-library count file for the mapped barcodes.
dna_representation_count_sum: Sum of supplied counts across input and all six plasmid DNA condition files for the mapped barcodes.
plasmid_N1_count: Raw/supplied plasmid DNA count in p53-high replicate 1 (WT TP53 plus Nutlin-3a).
plasmid_N2_count: Raw/supplied plasmid DNA count in p53-high replicate 2 (WT TP53 plus Nutlin-3a).
plasmid_WT1_count: Raw/supplied plasmid DNA count in untreated MCF7 WT replicate 1.
plasmid_WT2_count: Raw/supplied plasmid DNA count in untreated MCF7 WT replicate 2.
plasmid_KD1_count: Raw/supplied plasmid DNA count in p53-low stable TP53-knockdown replicate 1.
plasmid_KD2_count: Raw/supplied plasmid DNA count in p53-low stable TP53-knockdown replicate 2.
cDNA_N1_count: Raw/supplied reporter cDNA barcode count in p53-high replicate 1.
cDNA_N2_count: Raw/supplied reporter cDNA barcode count in p53-high replicate 2.
cDNA_KD1_count: Raw/supplied reporter cDNA barcode count in p53-low replicate 1.
cDNA_KD2_count: Raw/supplied reporter cDNA barcode count in p53-low replicate 2.
cDNA_N1_cpm: Library-size-scaled cDNA count per million for p53-high replicate 1.
cDNA_N2_cpm: Library-size-scaled cDNA count per million for p53-high replicate 2.
cDNA_KD1_cpm: Library-size-scaled cDNA count per million for p53-low replicate 1.
cDNA_KD2_cpm: Library-size-scaled cDNA count per million for p53-low replicate 2.
log2_activity_p53_high_rep1: Re-derived log2 reporter activity for p53-high replicate 1, using cDNA CPM and summed DNA representation.
log2_activity_p53_high_rep2: Re-derived log2 reporter activity for p53-high replicate 2, using cDNA CPM and summed DNA representation.
log2_activity_p53_low_rep1: Re-derived log2 reporter activity for p53-low replicate 1, using cDNA CPM and summed DNA representation.
log2_activity_p53_low_rep2: Re-derived log2 reporter activity for p53-low replicate 2, using cDNA CPM and summed DNA representation.
log2fc_nutlin_vs_p53_low_rep1: Re-derived log2 fold change of p53-high versus p53-low activity for replicate 1.
log2fc_nutlin_vs_p53_low_rep2: Re-derived log2 fold change of p53-high versus p53-low activity for replicate 2.
log2fc_nutlin_vs_p53_low_mean: Mean of the two replicate-level p53-high versus p53-low log2 fold changes.
replicate_direction_consistent: Whether both replicate log2 fold changes have the same activation/repression direction.
effect_size_call_1_5_log2: Effect-size-only call using the paper's absolute 1.5 log2 fold-change threshold and replicate-direction concordance; not a significance call.

Quality control

The paper required exact 17-bp CHEQ barcodes, retained PacBio barcodes uniquely linked to one enhancer region, collapsed enhancer regions at identical genomic loci, removed cDNA records with zero counts in any of N1, N2, KD1, or KD2, and used library-size/barcode-abundance normalization followed by DESeq2 differential analysis. Reported replicate concordance was r2=0.99 for p53-high, r2=0.56 for p53-low, and r2=0.72 for induced activity. Package QC retained 21,457 uniquely mapped barcodes and 18,588 fragment loci after the four-cDNA-nonzero filter; entries without an unambiguous PacBio mapping or with any zero cDNA count are excluded.

Curation notes

The supplied GEO archives contain count summaries and PacBio barcode mappings, not the authors' DESeq2 p-values/adjusted p-values or sequence FASTA. log2_activity values are re-derived from library-size-scaled cDNA CPM and summed input-plus-plasmid DNA representation; log2fc values compare p53-high (Nutlin) with p53-low and are effect sizes only. effect_size_call_1_5_log2 applies the paper's 1.5-log2 threshold but is not an original DESeq2 result. Named target points and unlabeled captured genomic fragments are both retained; a blank end denotes the named-point representation in the mapping data.

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