Experiment / E9SY880PGEpisomal Plasmid MPRA

Transient HEK293T plasmid barcode screen

Molecular barcoding of viral vectors enables mapping and optimization of mRNA trans-splicing

A barcoded splice-acceptor plasmid library containing random Syn1 intron 9-10 fragments was co-transfected with the splice-donor plasmid in HEK293T cells. Targeted sequencing of trans-spliced cDNA 48 hours after transfection reports barcode-level splicing output.

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

The episomal acceptor library was generated from randomly fragmented mouse Syn1 intron 9-10 sequence, inserted in both orientations, and labeled with a 20-nt (VHDB)x5 barcode in the 3-prime UTR. HEK293T cells received the library and full-intron splice-donor plasmid by transient transfection; the functional readout is targeted Illumina sequencing of barcodes in trans-spliced mRNA.

Processed data

50 rows per page. Click a cell to inspect its full value.

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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 · 23 definitions
barcode
Unique 20-nt molecular barcode from the splice-acceptor construct.
fragment_id
Identifier for the mapped Syn1 intron 9-10 reference-supported fragment interval.
insert_orientation_relative_to_Syn1
Whether the inserted binding-domain sequence is in Syn1 transcript-sense or reverse-complement orientation.
fragment_start_in_target_transcript_1based
1-based start of the mapped segment in transcript-oriented Syn1 intron 9-10 sequence.
fragment_end_in_target_transcript_1based
1-based end of the mapped segment in transcript-oriented Syn1 intron 9-10 sequence.
genomic_start_GRCm39
1-based GRCm39 genomic start on chrX for the mapped segment.
genomic_end_GRCm39
1-based GRCm39 genomic end on chrX for the mapped segment.
fragment_length
Length in bp of the exact reference-supported mapped segment.
target_sequence_transcript_orientation
Reference sequence for the mapped segment in Syn1 transcript orientation.
fragment_sequence_insert_orientation
Reference sequence in the orientation present in the library insert.
pacbio_library_read_count
Number of valid PacBio CCS library reads carrying this barcode in the lookup extraction.
pacbio_mapping_call_count
Number of PacBio CCS reads contributing an accepted fragment mapping call for this barcode.
pacbio_mapping_agreement
Fraction of accepted PacBio mapping calls supporting the selected orientation.
pacbio_exact_match_length
Length in bp of the best contiguous exact PacBio-to-reference segment used for mapping.
dna_count
Barcode count in the DNA/library reference measurement used as the denominator.
rna_count
Barcode count in the targeted cDNA/RNA splicing measurement.
dna_cpm
DNA count per million normalized over all lookup barcodes with available DNA counts.
rna_cpm
RNA count per million normalized over all lookup barcodes with available RNA counts.
log2_rna_dna
Log2 RNA/DNA activity score using a 0.5 CPM pseudocount.
dna_count_source
Description of the DNA/library measurement used for dna_count.
dna_sra_accession
SRA run accession for the DNA/library measurement, or PacBio lookup source.
rna_sra_accession
SRA run accession for the RNA/splicing measurement.
qc_pass
True for rows retained after the package-level QC filters.

Quality control

The paper's workflow trimmed vector flanks, retained the designed 20-nt V-H-D-B barcode, required unique fragment alignment, and removed PCR template-switching artifacts. For this package, only exact 20-nt ACGT barcodes matching the PacBio CCS lookup and the designed (VHDB)x5 cycle were counted; PacBio mappings required a unique 17-mer seed, an in-range reference-supported exact segment of at least 30 bp, and at least 0.5 orientation-call agreement. Processed rows additionally require dna_count >= 1 and RNA count >= 10. There is one public sequencing run per screen condition, so replicate correlation, dispersion, and FDR filters are not available; failed rows were omitted.

Curation notes

This screen uses PacBio CCS plasmid-library barcode counts as the DNA/library proxy because the transient screen has no separate DNA reference run in PRJNA403798. The paper reports a 48-hour post-transfection readout and one public RNA run (SRR6019579). The generated table contains 12119 QC-passing barcode rows; library normalization totals are dna=54022 and rna=500862.

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