Experiment / E5YEREF8SEpisomal Plasmid MPRA

IFNB multi-hit sampling MPRA in Sendai-virus-induced and mock-treated HEK293T/17 cells

Systematic dissection and optimization of inducible enhancers in human cells using a massively parallel reporter assay

A multi-hit sampling library of randomized human IFNB enhancer variants was assayed in HEK293T/17 cells in paired Sendai-virus-induced and mock-infected states. Each variant was represented by one reporter tag and measured in one biological transfection replicate per state.

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Organism: Human
Taxonomy ID: NCBITaxon:9606
Biosample: CVCL:1926
Reference genome: NCBI36/hg18
Design focus: Variant-focused
Region of interest: Not reported / not applicable

Perturbation & assay details

Sendai virus infection at MOI 10 for 16 h (induced) versus mock infection for 16 h (uninduced)

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. Multi-hit sampling: approximately 27,000 IFNB variants, one tag per variant, assayed in one induced and one uninduced library. Sample accessions: GSM792103-GSM792106. The source activity ratios are author-provided normalized mRNA/plasmid tag ratios for induced and uninduced states.

Processed data

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Visible columns (25 of 25)

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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 · 25 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_sequence: The single 10-nt reporter tag associated with the variant.
dna_count_induced_rep1: Raw plasmid DNA count for the induced library.
rna_count_induced_rep1: Raw reporter mRNA count for the induced library.
activity_ratio_induced_rep1: Author-provided library-size-normalized mRNA/plasmid activity ratio for the induced library.
log2_activity_induced_rep1: Base-2 logarithm of the induced activity ratio.
dna_count_uninduced_rep1: Raw plasmid DNA count for the uninduced library.
rna_count_uninduced_rep1: Raw reporter mRNA count for the uninduced library.
activity_ratio_uninduced_rep1: Author-provided library-size-normalized mRNA/plasmid activity ratio for the uninduced library.
log2_activity_uninduced_rep1: Base-2 logarithm of the uninduced activity ratio. Blank when the source ratio is zero and the log2-derived quantity is undefined.
mean_log2_activity_induced: The single induced replicate log2 activity value.
mean_log2_activity_uninduced: The single uninduced replicate log2 activity value. Blank when the source ratio is zero and the log2-derived quantity is undefined.
log2_inducibility: Induced log2 activity minus uninduced log2 activity. Blank when the source ratio is zero and the log2-derived quantity is undefined.
induced_over_uninduced_fold_change: 2 raised to log2_inducibility; induced-to-uninduced activity fold change. Blank when the source ratio is zero and the log2-derived quantity is undefined.
wt_normalized_log2_activity_induced: Induced log2 activity minus the mean across hINFB1_enhancer wild-type controls.
wt_normalized_log2_activity_uninduced: Uninduced log2 activity minus the mean across hINFB1_enhancer wild-type controls. Blank when the source ratio is zero and the log2-derived quantity is undefined.
wt_normalized_log2_inducibility: Variant log2 inducibility minus wild-type-control mean log2 inducibility. Blank when the source ratio is zero and the log2-derived quantity is undefined.
qc_pass: True for rows present in the author ratio worksheet and passing finite, nonnegative source-ratio validation; zero ratios are retained and their undefined log2-derived fields are blank.

Quality control

The paper required exact tag/construct matching, discarded tags with plasmid counts below 20 in every sequenced plasmid pool, normalized mRNA/plasmid ratios by total plasmid-to-mRNA tag counts, and used permutation tests with 5% FDR for information footprints. Package QC retained 26933 of 26933 rows available in the corresponding author ratio worksheet and excluded 0 rows. The source-valid randmut_16768 row has a zero uninduced RNA/DNA ratio; it is retained, while log2-derived fields that require a positive ratio are left blank.

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.

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