A single-hit scanning library of synthetic cAMP-responsive enhancer (CRE) variants was assayed in HEK293T/17 cells after forskolin induction. Each variant was represented by multiple 10-nt reporter tags, with two biological transfection replicates.
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Organism
Human
Taxonomy ID
NCBITaxon:9606
Biosample
CVCL:1926
Reference genome
Not reported / not applicable
Design focus
Variant-focused
Region of interest
Not reported / not applicable
Perturbation & assay details
100 uM forskolin for 5 h (induced state)
The library used 142-mer oligonucleotides containing an 87-nt variable enhancer sequence, a 10-nt identifying reporter tag, and invariant cloning sequences. Variants were cloned upstream of a minimal TATA-box promoter and luc2 ORF in a plasmid MPRA vector, transfected into HEK293T/17 cells, and quantified by 36-nt single-end Illumina Tag-Seq of reporter mRNA and plasmid DNA. Single-hit scanning: approximately 1,000 CRE variants; 13 tags per variant before plasmid-count filtering; 65 wild-type enhancer-tag pairs in the deposited count files. Sample accessions: GSM792099, GSM792100, GSM792101, GSM792102. The source activity ratios are author-provided median normalized mRNA/plasmid tag ratios for the induced state.
Processed data
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Column dictionary · 24 definitions
variant_id
Unique variant identifier assigned in the deposited library.
sequence
The 87-nt variable enhancer sequence from the deposited variant library.
variant_class
Library design class inferred from the deposited variant identifier.
design_position_0based
0-based scanning start position or split-scan offset encoded in the variant identifier; blank for controls/random variants.
designed_alt_base
Designed alternate base for single-substitution variants; blank for other designs.
sequence_length
Length of the deposited variable sequence in nucleotides; insertion variants are longer than 87 nt.
n_substitutions_vs_reference
Number of same-length nucleotide mismatches relative to the library wild-type sequence; blank for insertion variants.
changed_positions_0based
Comma-separated 0-based positions differing from the same-length wild-type sequence; blank for insertion variants.
tag_sequences
Semicolon-separated 10-nt reporter tags retained after the paper plasmid-count filter.
n_valid_tags_rep1
Number of retained reporter tags contributing to the replicate-1 median activity ratio.
n_valid_tags_rep2
Number of retained reporter tags contributing to the replicate-2 median activity ratio.
dna_count_sum_rep1
Sum of raw plasmid DNA tag counts over retained tags in replicate 1.
rna_count_sum_rep1
Sum of raw reporter mRNA tag counts over retained tags in replicate 1.
activity_ratio_rep1
Author-provided median library-size-normalized mRNA/plasmid activity ratio in induced replicate 1.
log2_activity_rep1
Base-2 logarithm of the author-provided induced replicate-1 activity ratio.
dna_count_sum_rep2
Sum of raw plasmid DNA tag counts over retained tags in replicate 2.
rna_count_sum_rep2
Sum of raw reporter mRNA tag counts over retained tags in replicate 2.
activity_ratio_rep2
Author-provided median library-size-normalized mRNA/plasmid activity ratio in induced replicate 2.
log2_activity_rep2
Base-2 logarithm of the author-provided induced replicate-2 activity ratio.
mean_log2_activity
Arithmetic mean of the two replicate log2 activity values.
wt_normalized_log2_activity
Mean log2 activity minus the mean across deposited synCRE_Promega wild-type controls.
published_pvalue_rep1
Author-provided Mann-Whitney p-value versus wild-type for induced replicate 1.
published_pvalue_rep2
Author-provided Mann-Whitney p-value versus wild-type for induced replicate 2.
qc_pass
True for rows retained after source plasmid-count and finite-activity QC.
Quality control
The paper required exact tag/construct matching, discarded tags with plasmid counts below 20 in every relevant plasmid pool, normalized mRNA/plasmid ratios by total plasmid-to-mRNA tag counts, and used Mann-Whitney tests with 5% FDR for single-hit variant comparisons. Package QC retained 999 of 999 rows available in the corresponding author ratio worksheet and excluded 0 rows; the retained rows have finite positive activity ratios and at least five valid reporter tags for single-hit libraries.
Curation notes
The processed activity_ratio columns are taken from the author-provided Supplementary Table ratio worksheets so that the reported plasmid-count filtering and total-count normalization are preserved. Raw DNA/RNA count summaries and tag sequences are joined from the GEO GSE31982 count archive. WT-normalized values use the mean of the deposited wild-type-control rows (synCRE_Promega_* or hINFB1_enhancer_*). CRE is synthetic and has no genomic coordinate; the IFNB variable sequence was derived from NCBI36/hg18, but the paper does not provide an exact coordinate for this 87-nt tested fragment. The single-hit libraries contain induced-state data only; paired inducibility is available in the multi-hit tables.