HEK293T episomal MPRA of downstream-of-PAS 8-bp sequence variants
Slight Variations in the Sequence Downstream of the Polyadenylation Signal Significantly Increase Transgene Expression in HEK293T and CHO CellsNine barcoded plasmid libraries tested randomized sequence variants in overlapping 8-bp windows located +17 to +56 bp downstream of the polyadenylation signal of an eGFP reporter in transiently transfected HEK293T cells. The processed table aggregates the authors' barcode-level measurements to one ROI/mutation row per library and preserves the associated barcode count and activity dispersion.
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
Basal / Untreated
Episomal pTTC-hPGK-eGFP plasmid libraries placed randomized 18-bp barcodes in the reporter 3' region and randomized 8-bp ROIs at nine overlapping positions downstream of the PAS. An equimolar pool of two wildtype reference constructs with specific 20-bp barcodes was spiked into each library at 1:100. HEK293T cells were harvested 48 h after transfection; mapping, plasmid-normalization, and cDNA-expression samples were sequenced as 151-nt single-end Illumina MiSeq reads and decoded with MPRAdecoder. Barcode RNA/DNA abundance was normalized to the wildtype reference, and ROI activity was averaged across associated barcodes. The paper additionally validated selected sequences with non-barcoded eGFP and NanoLuc constructs in HEK293T and CHO cells, but those validation assays are not part of this MPRA table.
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 · 26 definitions
- library_id
- Overlapping ROI window relative to the PAS, formatted as +start..+end bp.
- window_start_bp_downstream_of_PAS
- Start coordinate of the library window in bp downstream of the PAS.
- window_end_bp_downstream_of_PAS
- End coordinate of the library window in bp downstream of the PAS.
- roi_sequence
- Observed sequence of the region of interest in the source MPRAdecoder output.
- roi_length_bp
- Length in bp of the observed ROI sequence.
- barcode_count
- Number of source barcode rows associated with this ROI.
- barcode_length_min_bp
- Shortest observed barcode length among associated barcode rows.
- barcode_length_max_bp
- Longest observed barcode length among associated barcode rows.
- barcodes_with_nonzero_expression
- Associated barcode rows with a positive expression mean.
- barcodes_with_zero_expression
- Associated barcode rows with zero expression mean.
- normalization_replicate_count
- Number of normalization measurement columns in the source workbook.
- expression_replicate_count
- Number of expression measurement columns in the source workbook.
- normalization_mean_across_barcodes
- Arithmetic mean of the source per-barcode normalization means.
- expression_mean_across_barcodes
- Arithmetic mean of the source per-barcode expression means.
- normalized_expression_mean_across_barcodes
- Arithmetic mean of source normalized expression values before wildtype-reference scaling.
- normalized_expression_sd_across_barcodes
- Sample standard deviation of source normalized expression values across associated barcodes.
- activity_ratio_source_column
- Original source column used for the wildtype-reference-normalized activity ratio.
- activity_ratio_to_wildtype_reference
- Arithmetic mean of the source activity ratios across associated barcodes; 1 is the wildtype-reference level.
- activity_ratio_median_across_barcodes
- Median source activity ratio across associated barcodes.
- activity_ratio_sd_across_barcodes
- Sample standard deviation of source activity ratios across associated barcodes.
- activity_ratio_min
- Minimum source activity ratio among associated barcodes.
- activity_ratio_max
- Maximum source activity ratio among associated barcodes.
- log2_activity_ratio
- Log2 of the mutation-level activity ratio to the wildtype reference.
- effect_category
- Mutation-level effect call: increased_2x_or_more, decreased_2x_or_more, or within_2x.
- qc_pass
- Boolean indicating that the ROI group passed package QC and was retained.
- source_file
- Raw GEO workbook from which this ROI group was processed.
Quality control
The source spreadsheets are MPRAdecoder processed output, with barcode-ROI associations recovered from mapping samples and barcode expression normalized to plasmid abundance. Package QC retained 226,964 of 227,755 published ROI/mutation groups: groups were required to have finite values, a positive normalization mean, and at least one nonzero expression signal across the expression replicates; the 791 groups with zero total expression across both expression replicates (the groups counted as zero-expression in published Table 2) were excluded. Groups with some zero-expression barcode rows were retained when other associated barcodes provided expression. Source-reported ROI length variants of 7 or 9 bp were retained because they are present in the authors' processed output and are flagged in the table.
Curation notes
This is one MPRA screen comprising nine overlapping synthetic libraries, not nine independent biological conditions. The raw workbooks contain one row per barcode (406,137 rows); the processed table uses one row per unique ROI/mutation (226,964 retained rows) because the paper's reported activity and Table 2 summaries are ROI-level averages across associated barcodes. The nominal design is 8-bp ROI and 18-bp barcode, but the source output includes 7/9-bp ROI and 16-20-bp barcode observations, consistent with the decoder's allowed length tolerance; these observations were retained and their lengths are explicit. No genomic locus or reference assembly applies to the synthetic reporter library. HEK293T was resolved to Cellosaurus CVCL:0063. CHO and selected non-barcoded reporter validations are described in the paper but are not separate MPRA experiments.