Barcoded tetO2 variant MPRA with TetR and doxycycline
High-Throughput Characterization of Tetracycline Repressor Function on Tetracycline Operator 2 VariantsAn episomal pcDNA3.1-tetO-BC-mGL library containing 96 designed tetO2 operators and approximately 100 random 24-nt barcodes per operator was assayed in HEK293 cells. Variant-level TetR repression was measured by comparing RNA/DNA reporter activity with TetR present under untreated versus doxycycline-treated conditions, with a mGreenLantern co-transfection arm as a repressor-independent control.
Processed tables are specific to each experiment. Column names, units, measurements, and table structure are not standardized across the database. Check this experiment’s column definitions and quality-control notes before comparing or combining data.
Perturbation & assay details
TetR co-transfection (pCAGTetRnls) with 20 µg/mL doxycycline for approximately 24 h versus untreated; a pcDNA3.1-mGreenLantern co-transfection arm served as a control
Standard episomal plasmid MPRA with a CMV-driven mGreenLantern reporter and random 24-nt 5′-UTR barcodes. The library contained tetO2 operator variants inserted downstream of CMV; barcode/variant dictionaries were generated by AVITI 2×75 sequencing, RNA barcode libraries were sequenced after DNase treatment and RT-PCR, and MPRAnalyze-derived comparisons were used to calculate TetR repression. Aggregate Table S2 values are retained because barcode-level DNA/RNA counts and replicate matrices were not deposited.
Processed data
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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 · 16 definitions
- element_id
- Operator or control name from Supplementary Table S2
- variant_class
- Normalized class derived from the Table S1 type field
- source_variant_type
- Original Table S1 type label
- operator_sequence_5to3
- 5′-to-3′ DNA sequence of the tested operator/control from Table S1
- sequence_length
- Length of operator/control sequence in nucleotides
- mutation_positions
- 1-based positions differing from the canonical tetO2 x 1 sequence for mutagenized rows; comma-separated
- reference_bases
- Canonical tetO2 x 1 bases at mutation positions; comma-separated
- alternate_bases
- Bases present in the tested sequence at mutation positions; comma-separated
- mutations_vs_tetO2_x1
- Compact mutation notation position:reference>alternate relative to tetO2 x 1
- tetracycline_repressor_repression_pct
- TetR repression percentage reported in Supplementary Table S2
- mgreenlantern_control_repression_pct
- mGreenLantern control repression percentage reported in Supplementary Table S2
- repression_rank
- Descending rank by TetR repression percentage across retained Table S2 rows
- is_positive_control
- Whether the element is labeled as a positive control in Table S1
- is_negative_control
- Whether the element is labeled as a negative control in Table S1
- qc_status
- Packaging QC status; all retained rows passed
- source_table
- Supplementary table provenance for the row
Quality control
The authors filtered barcode/variant dictionary reads by variant-region quality (at least 30, excluding the first nucleotide), barcode-region quality (at least 25 after the first 7 nucleotides and greater than 10 within the first 7), and barcode-to-variant mapping. Ambiguous mappings were retained only when the most frequent pairing exceeded the second pairing by more than 100-fold; Starcode clustering at Levenshtein distance 1 applied the same 100-fold centroid criterion. MPRA sample depth was normalized using negative controls and TetR repression was calculated from negative-control-normalized RNA/DNA activity. For packaging, only Table S2 rows with numeric TetR and mGreenLantern values and an exact sequence/type match to Table S1 were retained: 93 rows. The three Table S1 entries explicitly marked as not found in the variant library (tetO8G, tetO11C, randtetO8) were not included.
Curation notes
The public data are aggregate variant-level repression percentages rather than raw barcode-level counts, DNA/RNA libraries, or replicate-level statistics. The processed table intentionally retains positive and negative controls to preserve the assay's repression scale. Mutation annotations are computed from the deposited sequences against tetO2 x 1; controls and the tandem tetO2 x 2 positive control have blank mutation fields when a direct comparison is not appropriate. The final article body is closed access; full_text.txt contains the complete abstract and the complete freely available supporting-information Methods text, while the original SI PDF and XLSX are preserved in raw_data.