Experiment / E7KZTOXCPTargeted Genomic Integration MPRA

QeFS in twi:CD2+ embryonic mesoderm

Quantitative-enhancer-FACS-seq (QeFS) reveals epistatic interactions among motifs within transcriptional enhancers in developing Drosophila tissue

A pooled library of wild-type and motif-mutated Drosophila enhancers was genomically integrated at a common landing site and assayed in stage 11–12 embryos crossed to a twi:CD2 driver. The CD2-positive whole-mesoderm fraction was quantified by targeted reporter RNA and DNA sequencing with UMI-tagged reporter molecules.

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Organism: Fruit fly
Taxonomy ID: NCBITaxon:7227
Biosample: UBERON:0000926
Reference genome: Not reported / not applicable
Design focus: Synthetic / Motif-focused
Region of interest: Not reported / not applicable

Perturbation & assay details

Wild-type versus systematic single, pairwise, and three-way mutations of Deaf1, Schlank, ZIPIC, and/or CG12236-PB binding-site motifs; Act57B and β3Tub60D positive-control constructs were included.

Quantitative enhancer-FACS-Seq (QeFS) uses site-specific φC31 genomic integration in Drosophila, 8-mer enhancer barcodes, and degenerate 20-mer tags in the reporter 3′ UTR. FACS separates the CD2-marked cell population rather than binning reporter fluorescence; targeted RNA/DNA UMI counts calculate DNA-normalized reporter activity across three twi:CD2+ collection days.

Processed data

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Visible columns (28 of 28)

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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 · 28 definitions

element_id: Canonical construct identifier from Additional file 3/Table S3; GEO's ZIPIC token 7/71 is normalized to Z/Z1.
source_construct_name: Construct identifier exactly as reported in the GEO fullFiltered source table.
parent_enhancer: Parent endogenous enhancer or positive-control element.
element_type: Whether the element is an endogenous enhancer construct or a positive-control construct.
variant_class: Wild-type, single-motif, pairwise-motif, three-way-motif, or positive-control class.
mutated_motifs: Semicolon-delimited motifs or control sites mutated relative to the corresponding wild type; none for wild type.
barcoded_sequence: Representative tested sequence from Additional file 3/Table S3, including the flanking enhancer barcodes.
sequence_length_bp: Length of barcoded_sequence in base pairs.
left_barcode_8mer: First 8 bases of the representative sequence-table barcode.
right_barcode_8mer: Last 8 bases of the representative sequence-table barcode.
n_measurements: Number of qualifying tag-by-collection measurements; corresponds to the author numTags summary field.
n_unique_reporter_tags: Number of unique degenerate 20-mer reporter tags among qualifying measurements.
n_collection_replicates: Number of distinct collection-day labels represented in qualifying measurements.
tag_ids: Semicolon-delimited unique 20-mer reporter tags represented for the construct.
dna_umi_sum: Sum of DNA UMI counts across qualifying measurements.
rna_umi_sum: Sum of RNA UMI counts across qualifying measurements.
mean_norm_dna: Mean normalized DNA UMI abundance across qualifying measurements.
mean_norm_rna: Mean normalized RNA UMI abundance across qualifying measurements.
total_activity_rna_dna: Sum of per-measurement RNA/DNA normalized activity values.
mean_activity_rna_dna: Mean DNA-normalized reporter activity (normUMI; RNA UMI/DNA UMI after library-size normalization).
sd_activity_rna_dna: Sample standard deviation of per-measurement normalized reporter activity.
sem_activity_rna_dna: Standard error of the mean normalized reporter activity.
wildtype_mean_activity: Mean activity of the corresponding parent wild-type construct in this experiment; null when no passing wild type was measured.
fold_change_vs_wt: Mean activity divided by the corresponding wild-type mean activity; null when no passing wild type was available.
log2_fold_change_vs_wt: Base-2 logarithm of fold_change_vs_wt; null when no passing wild type was available.
ci_t_vs_wt: Conover–Iman t statistic for the pairwise comparison with the corresponding wild type.
adjusted_p_vs_wt: Benjamini–Hochberg-adjusted Conover–Iman p-value versus the corresponding wild type.
significant_vs_wt: yes/no significance call from the authors' adjusted pairwise test; blank for wild type or unavailable comparison.

Quality control

Applied the authors' stated filters to the GEO fullFiltered per-tag tables: removed individual measurements with fewer than 65 DNA UMIs and retained construct groups with at least 4 qualifying tag-level measurements. The resulting construct-level means, standard deviations, totals, and standard errors were cross-checked against Additional file 4/Table S4; only constructs passing these filters are present.

Curation notes

The table contains 86 QC-passing constructs and is aggregated from the GEO TWICD2PLUS fullFiltered per-tag measurements. The sequence is the representative sequence-table record rather than every barcode-linked reporter molecule; all qualifying reporter tags remain available in raw_data. The ChIPCRM5792 series has no QC-passing WT construct, so WT-relative fields are null for that parent.

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