Transient plasmid MPRA of the shared library of 5,418 145-bp human enhancer constructs, including wild-type sequences and engineered regulatory-motif variants, in HepG2 cells. Reporter RNA was quantified from ten 10-nt barcode tags per construct in two biological replicates and normalized to two plasmid-library replicates.
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Organism
Human
Taxonomy ID
NCBITaxon:9606
Biosample
CVCL:0027
Reference genome
hg19
Design focus
Synthetic / Motif-focused
Region of interest
Not reported / not applicable
Perturbation & assay details
Basal / Untreated
A 145-bp enhancer sequence was placed upstream of an SV40 early enhancer/promoter and luc2 reporter; each construct carried ten distinct 10-nt tags. The plasmid pool was transiently transfected into HepG2 cells, and barcode counts from poly(A)+ reporter RNA were compared with counts from the plasmid library. The library contains natural human enhancer sequences and synthetic motif perturbations rather than natural SNP alleles.
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Column dictionary · 19 definitions
construct_id
GEO construct identifier (PK_construct_*).
sequence_145bp
145-bp human enhancer or engineered motif-variant sequence in the reporter library.
barcode_count
Number of designed barcode tags associated with the construct (10).
plasmid_count_rep1
Total plasmid-library reads across the ten barcodes in plasmid replicate 1, before the per-barcode QC cutoff.
plasmid_count_rep2
Total plasmid-library reads across the ten barcodes in plasmid replicate 2, before the per-barcode QC cutoff.
rna_count_rep1
Total HepG2 reporter-RNA reads across the ten barcodes in mRNA replicate 1.
rna_count_rep2
Total HepG2 reporter-RNA reads across the ten barcodes in mRNA replicate 2.
n_qc_barcodes_rep1
Barcode observations in replicate 1 with plasmid count at least 40.
n_qc_barcodes_rep2
Barcode observations in replicate 2 with plasmid count at least 40.
n_qc_barcodes_total
Total barcode observations passing the plasmid-count cutoff across both replicates.
n_replicates_qc
Number of replicate-specific RNA/plasmid ratios available after QC (1 or 2).
rna_dna_ratio_rep1
Mean per-barcode RNA/plasmid ratio after library-size normalization for replicate 1, using QC-passing barcodes.
rna_dna_ratio_rep2
Mean per-barcode RNA/plasmid ratio after library-size normalization for replicate 2, using QC-passing barcodes.
rna_dna_ratio_mean
Mean of the available replicate-specific normalized RNA/plasmid ratios; canonical pre-log activity measure.
activity_log2_rep1
Replicate 1 log2 activity relative to the median canonical RNA/plasmid ratio across retained constructs.
activity_log2_rep2
Replicate 2 log2 activity relative to the median canonical RNA/plasmid ratio across retained constructs.
activity_log2_mean
Canonical construct activity, log2(mean normalized RNA/plasmid ratio divided by the median); zero-RNA values are represented as -7.
activity_log2_sd
Standard deviation across the available replicate-level log2 activities.
zero_rna_replacement
Whether the canonical RNA/plasmid ratio was zero and the activity score was replaced with -7.
Quality control
The deposited count data represent reads that matched a designed barcode and the expected upstream MPRA construct sequence. Following the publication's processing, counts were library-size normalized, RNA/plasmid ratios were computed per barcode, and barcode observations with plasmid counts below 40 were excluded. Construct activity was the mean of available replicate-specific barcode means, expressed as log2(activity divided by the median across retained constructs); zero-RNA activity values were set to -7. Of 5,418 deposited constructs, 5,372 retained at least one QC-passing barcode observation (89,882 of 108,360 barcode-replicate observations); 46 constructs with no retained barcode were excluded from this table. 5,358 retained constructs had both replicate ratios and 14 had one. The replicate Pearson correlation recomputed from retained construct ratios was 0.690, consistent with the paper's reported HepG2 value of 0.69.
Curation notes
The same plasmid library is used for both HepG2 and K562 experiments. Plasmid replicate 1 was paired with mRNA replicate 1 and plasmid replicate 2 with mRNA replicate 2 for replicate-specific ratios. GEO provides construct sequences, ten-barcode lists, and count vectors but not the paper's Supplemental Data S1 annotations for motif/factor, wild-type versus engineered variant, or genomic coordinates; those labels were not inferred. The article notes that HepG2 is readily transfected and reports a HepG2 replicate correlation of 0.69. The reference assembly is recorded as hg19 based on the ENCODE-era source context; the main article text does not explicitly name an assembly.