LibSeq 7-mer reporter screen of RNase H oligonucleotide specificity in HeLa cells
Dissecting the target specificity of RNase H recruiting oligonucleotides using massively parallel reporter analysis of short RNA motifsA synthetic 7-mer library embedded in a fixed HAND2-derived 3′ UTR context was transfected into HeLa cells to measure endogenous post-transcriptional regulation and RNase H-recruiting oligonucleotide effects. The single processed table joins plasmid and untreated transfection counts with five parallel LNA-gapmer cotransfection conditions and sequence-level binding annotations.
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
25 nM RNase H-recruiting LNA-modified phosphorothioate oligonucleotide A, B, B-4DNA, B-3DNA, or B-1DNA; matched no-oligonucleotide transfection controls.
LibSeq uses an episomal pGL4.12-TK-MCS+ luciferase reporter library with a seven-base degenerate sequence in the 3′ UTR, keeping the flanking reporter context constant. HeLa cells received 6.3 µg library plasmid; treatment samples also received 450 pmol oligonucleotide (25 nM). After 24 h, poly(A)+ RNA was DNase treated, reverse transcribed with a LibSeq-specific primer, PCR amplified, and sequenced on an Illumina HiSeq 2000. Reporter 7-mer abundance was counted from plasmid DNA and cDNA libraries; the table contains the untreated control and five oligonucleotide cotransfections.
Processed data
50 rows per page. Click a cell to inspect its full value.
Visible columns (56 of 56)
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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 · 56 definitions
- element_id
- Stable package identifier formed from the LibSeq 7-mer sequence.
- seven_mer
- The seven-base degenerate reporter motif in the source-table orientation.
- plasmid_rep1_count
- Raw grouped sequencing count for plasmid library replicate 1.
- plasmid_rep2_count
- Raw grouped sequencing count for plasmid library replicate 2.
- untreated_control_rep1A_count
- Raw grouped count for untreated transfection replicate 1A, the first technical library-preparation replicate.
- untreated_control_rep1B_count
- Raw grouped count for untreated transfection replicate 1B, the second technical library-preparation replicate.
- untreated_control_rep1_count
- Raw grouped count for untreated transfection replicate 1; the source value equals replicate 1A plus 1B and is the matched control used for oligonucleotide fold-change calculations.
- untreated_control_rep2_count
- Raw grouped count for untreated transfection biological replicate 2.
- untreated_control_rep3_count
- Raw grouped count for untreated transfection biological replicate 3.
- oligo_A_count
- Raw grouped cDNA count after cotransfection with RNase H-recruiting oligonucleotide A.
- oligo_B_count
- Raw grouped cDNA count after cotransfection with RNase H-recruiting oligonucleotide B.
- oligo_B_4DNA_count
- Raw grouped cDNA count after cotransfection with B-4DNA, the shortened central-DNA-gap version of B.
- oligo_B_3DNA_count
- Raw grouped cDNA count after cotransfection with B-3DNA.
- oligo_B_1DNA_count
- Raw grouped cDNA count after cotransfection with B-1DNA.
- plasmid_rep1_rmr
- Plasmid replicate 1 count normalized to the complete source-column total and expressed as reads per million reads (RMR).
- plasmid_rep2_rmr
- Plasmid replicate 2 count normalized to the complete source-column total and expressed as RMR.
- untreated_control_rep1A_rmr
- Untreated control replicate 1A count normalized to its complete source-column total and expressed as RMR.
- untreated_control_rep1B_rmr
- Untreated control replicate 1B count normalized to its complete source-column total and expressed as RMR.
- untreated_control_rep1_rmr
- Matched untreated control replicate 1 count normalized to its complete source-column total and expressed as RMR.
- untreated_control_rep2_rmr
- Untreated control replicate 2 count normalized to its complete source-column total and expressed as RMR.
- untreated_control_rep3_rmr
- Untreated control replicate 3 count normalized to its complete source-column total and expressed as RMR.
- oligo_A_rmr
- Oligonucleotide A cDNA count normalized to its complete source-column total and expressed as RMR.
- oligo_B_rmr
- Oligonucleotide B cDNA count normalized to its complete source-column total and expressed as RMR.
- oligo_B_4DNA_rmr
- B-4DNA cDNA count normalized to its complete source-column total and expressed as RMR.
- oligo_B_3DNA_rmr
- B-3DNA cDNA count normalized to its complete source-column total and expressed as RMR.
- oligo_B_1DNA_rmr
- B-1DNA cDNA count normalized to its complete source-column total and expressed as RMR.
- plasmid_mean_rmr
- Mean of the two normalized plasmid-library RMR values.
- untreated_control_mean_rmr
- Mean of normalized untreated transfection replicate 1, 2, and 3 RMR values.
- endogenous_log2_cDNA_vs_plasmid_mean
- Derived log2 ratio of mean untreated transfection RMR to mean plasmid RMR; a negative value indicates lower reporter abundance after transfection and is not a published significance statistic.
- oligo_A_log2_fold_change_vs_untreated
- Derived log2 ratio of oligonucleotide A RMR to matched untreated control replicate 1 RMR; negative values indicate repression and blank means the treatment count was zero.
- oligo_B_log2_fold_change_vs_untreated
- Derived log2 ratio of oligonucleotide B RMR to matched untreated control replicate 1 RMR; negative values indicate repression and blank means the treatment count was zero.
- oligo_B_4DNA_log2_fold_change_vs_untreated
- Derived log2 ratio of B-4DNA RMR to matched untreated control replicate 1 RMR; negative values indicate repression and blank means the treatment count was zero.
- oligo_B_3DNA_log2_fold_change_vs_untreated
- Derived log2 ratio of B-3DNA RMR to matched untreated control replicate 1 RMR; negative values indicate repression and blank means the treatment count was zero.
- oligo_B_1DNA_log2_fold_change_vs_untreated
- Derived log2 ratio of B-1DNA RMR to matched untreated control replicate 1 RMR; negative values indicate repression and blank means the treatment count was zero.
- oligo_A_at_least_2fold_repressed
- TRUE when the A treatment is at or below half the matched untreated RMR, or has zero observed reads.
- oligo_B_at_least_2fold_repressed
- TRUE when the B treatment is at or below half the matched untreated RMR, or has zero observed reads.
- oligo_B_4DNA_at_least_2fold_repressed
- TRUE when the B-4DNA treatment is at or below half the matched untreated RMR, or has zero observed reads.
- oligo_B_3DNA_at_least_2fold_repressed
- TRUE when the B-3DNA treatment is at or below half the matched untreated RMR, or has zero observed reads.
- oligo_B_1DNA_at_least_2fold_repressed
- TRUE when the B-1DNA treatment is at or below half the matched untreated RMR, or has zero observed reads.
- oligo_A_binding_score
- Supplementary Table S2 Needleman–Wunsch binding score for oligonucleotide A against the reporter target; Watson–Crick pairs score 1 and the maximum is 13.
- oligo_A_rna_rna_dG_kcal_mol
- Supplementary Table S2 estimated RNA/RNA duplex standard free energy for oligonucleotide A and the target, in kcal/mol; the paper notes this is an approximation for LNA-modified chemistry.
- oligo_B_binding_score
- Supplementary Table S2 Needleman–Wunsch binding score for oligonucleotide B against the reporter target.
- oligo_B_rna_rna_dG_kcal_mol
- Supplementary Table S2 estimated RNA/RNA duplex standard free energy for oligonucleotide B and the target, in kcal/mol.
- oligo_A_supplementary_selected
- TRUE when the motif appears in the selected-target list of Supplementary Table S3; FALSE otherwise.
- oligo_A_wobble
- Reported oligonucleotide A:RNA wobble notation from Supplementary Table S3, when annotated.
- oligo_A_wobble_note
- Comparison or positional note supplied in Supplementary Table S3 for the A selected-target annotation.
- oligo_B_supplementary_selected
- TRUE when the motif appears in the selected-target list of Supplementary Table S4; FALSE otherwise.
- oligo_B_wobble
- Reported oligonucleotide B:RNA wobble notation from Supplementary Table S4, when annotated.
- oligo_B_wobble_note
- Comparison or positional note supplied in Supplementary Table S4 for the B selected-target annotation.
- supplementary_table_5_b_log2_fold_change
- Published B fold-change reduction (log2) for the selected mismatch/bulge comparison set in Supplementary Table S5; blank outside that set.
- supplementary_table_5_b4dna_log2_fold_change
- Published B-4DNA fold-change reduction (log2) for the selected comparison set in Supplementary Table S5; blank outside that set.
- supplementary_table_5_reference_rmr
- Published untreated transfection RMR for the selected comparison set in Supplementary Table S5; blank outside that set.
- b_b4dna_comparison_effect
- Supplementary Table S5 classification of whether the motif was affected by both treatments or had the largest effect with B.
- b_mismatch_position
- Reported mismatch position in the B/B-4DNA duplex alignment, when present in Supplementary Table S5.
- b_rna_bulge_position
- Reported RNA bulge position in the B/B-4DNA duplex alignment, when present in Supplementary Table S5.
- qc_pass
- TRUE for every row retained after the package-level sequence and coverage QC filter.
Quality control
The paper trimmed 20 nt from read 3′ ends, discarded reads with more than 10% of positions below Q20, clipped the expected library sequences, removed reads lacking clipping sequences, failed base calls, or the required length, reverse complemented and grouped exact 7-mers, and required no product in minus-RT controls. The authors excluded contaminated 7-mers GGGCCAA and ACATACG from their endogenous analysis; they are already absent from Supplementary Table S1. Package QC retained only exact A/C/G/T 7-mers with at least one count in both plasmid replicates and in the matched untreated transfection replicate 1, retaining 16,327 of 16,382 source rows and excluding 55 low-coverage/dropout rows. Treatment zero counts were retained as below-detection observations; their treatment log2 fold-change field is blank because the ratio is undefined. RMR denominators use all 16,382 source rows so values reproduce the paper's selected-target tables.
Curation notes
The study uses the same episomal LibSeq reporter library across endogenous-control and RNase H oligonucleotide conditions, so it is represented as one integrated MPRA experiment rather than duplicating the same 7-mer rows into five treatment folders. The source oligonucleotides are A = TATcagctacTTT, B = TATtgaacgtTTT, B-4DNA = TATtgaAcgttTT, B-3DNA = TAttgAacgTtTT, and B-1DNA = TATtGaAcGtTtT; bold/uppercase bases are LNA and lowercase bases are DNA, all phosphorothioate-linked. Source Transfection replicate 1 is the sum of technical replicates 1A and 1B; the derived treatment log2 ratios use this matched control and reproduce the selected values in Supplementary Tables S3 and S4 to floating-point precision. The complete source spreadsheets are preserved in raw_data. The 55 excluded rows are low-coverage/dropout motifs with zero counts in at least one of the two plasmid replicates or the matched untreated control; zero treatment counts are retained as below-detection measurements. The paper does not specify a reference assembly, and the seven-base library is synthetic despite using a HAND2-derived flanking context, so reference_genome and region_of_interest are null. HeLa is represented by Cellosaurus CVCL:0030 and human by NCBITaxon:9606.