A 12,000-oligonucleotide episomal reporter library tested 1,200 95-bp variable regions derived from human p53 binding sites, motif-scrambled controls, and random synthetic controls in primary human fetal fibroblasts GM06170. Reporter activity was measured in untreated cells and after 500 nM doxorubicin treatment for 6 h, with two biological replicates per condition.
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Organism
Human
Taxonomy ID
NCBITaxon:9606
Biosample
CL:0000057
Reference genome
hg19
Design focus
Region-focused
Region of interest
Not reported / not applicable
Perturbation & assay details
Untreated vs 500 nM doxorubicin for 6 h
The library contained 5,700 oligos with endogenous sequences from 570 p53 binding sites, 5,700 matched oligos with scrambled p53 recognition motifs, and 600 oligos representing 60 random sequences. Each of the 1,200 variable regions was represented by ten independent 10-bp barcode oligos. For the promoter-proximal design, the variable sequence was cloned immediately upstream of a minimal promoter-GFP reporter; targeted RNA sequencing of GFP reporter barcodes provided the readout, normalized to the input MPRA vector pool. The GEO table reports the barcode-collapsed variable-region values for two untreated and two doxorubicin-treated samples.
Processed data
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Column dictionary · 20 definitions
element_id
Unique package identifier for the GEO variable-region record.
sequence
The 95-bp MPRA variable-region sequence, preserving the source capitalization used to mark motif and flanking sequence.
chromosome
GEO-reported chromosome for the genomic p53-site or scrambled control; blank for random synthetic controls.
start
GEO-reported start coordinate of the 95-bp genomic element; blank for random synthetic controls.
end
GEO-reported end coordinate of the 95-bp genomic element; blank for random synthetic controls.
coordinate_span
Reported genomic span calculated as end minus start plus one; blank for random synthetic controls.
oligo_type
Library category: Endogenous p53 Motif, Scrambled p53 Motif, or Random Sequence.
untreated_rep1_normalized_expression
Normalized reporter expression for untreated biological replicate 1.
untreated_rep2_normalized_expression
Normalized reporter expression for untreated biological replicate 2.
untreated_mean_normalized_expression
Mean normalized reporter expression across the two untreated replicates.
untreated_sem
Standard error of the mean for the untreated normalized expression across two replicates.
doxorubicin_rep1_normalized_expression
Normalized reporter expression after doxorubicin treatment for biological replicate 1.
doxorubicin_rep2_normalized_expression
Normalized reporter expression after doxorubicin treatment for biological replicate 2.
doxorubicin_mean_normalized_expression
Mean normalized reporter expression across the two doxorubicin-treated replicates.
doxorubicin_sem
Standard error of the mean for the doxorubicin-treated normalized expression across two replicates.
doxorubicin_vs_untreated_fold_change
Published doxorubicin-to-untreated reporter-expression fold change.
doxorubicin_vs_untreated_log2_fold_change
Log2 transformation of the published doxorubicin-to-untreated fold change.
adjusted_p_value
GEO-provided multiple-testing-adjusted p-value for the doxorubicin-versus-untreated comparison.
significant_fdr_0_05
Boolean conversion of the GEO Sig field; true indicates adjusted p-value below 0.05.
source_row
One-based row number in the corresponding GEO tab-delimited source file, including its header row.
Quality control
The authors retained only sequencing reads whose first 10 bases exactly matched a library tag and whose following 26 bases matched the expected reporter sequence, summed reads across the ten tags for each variable region, normalized expression to the input vector pool, and used DESeq2 for differential expression. Package QC required a 95-nt A/C/G/T sequence, a recognized oligo type, finite normalized-expression/fold-change/p-value fields, an adjusted p-value in [0, 1], and a valid Yes/No significance value. Genomic records were required to have a valid chromosome and a 95-bp reported coordinate span; random synthetic controls were expected to have NA coordinates. All 1,200 of 1,200 GEO records passed and were retained, including non-significant elements.
Curation notes
This is a region-focused enhancer screen rather than an allelic-variant assay. Coordinates are retained as reported by GEO and correspond to hg19; the 95-bp span was checked against the source start/end fields. The sequence field is the variable region only, not the full 150-mer oligo or barcode-containing construct. The table includes the 570 endogenous p53-site sequences, 570 scrambled-motif matched controls, and 60 random synthetic controls, and retains non-significant records for unbiased downstream analysis. GM06170 is a primary human fetal fibroblast culture and is therefore mapped to the fibroblast Cell Ontology term CL:0000057 rather than a Cellosaurus immortalized-cell-line identifier.