Experiment / E704876BDEpisomal Plasmid MPRA

Untreated Jurkat allelic enhancer MPRA

Global discovery of lupus genetic risk variant allelic enhancer activity

The study's allele-focused MPRA library was transiently electroporated into the human Jurkat T-cell line in five independent biological replicates under basal conditions. Reporter DNA and RNA barcode counts were used to quantify allele-linked enhancer activity, with published Student's t-test comparisons for enVars.

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Organism: Human
Taxonomy ID: NCBITaxon:9606
Biosample: CVCL:0367
Reference genome: hg19
Design focus: Variant-focused
Region of interest: Not reported / not applicable

Perturbation & assay details

Basal / Untreated

The episomal pGL4.23-derived reporter placed allele-centered oligos upstream of a minimal promoter and eGFP reporter, with random 20-bp barcodes linking oligos to reporter transcripts. The source workbook contains nominally 200-bp synthesized oligo records (the allele-centered genomic insert plus cloning-flank sequence); indel records can be shorter, and forward-strand records are retained in the table. Jurkat cells were electroporated with the library in five independent replicates, recovered for 24 h, and collected for matched DNA and RNA barcode sequencing. DESeq2 compared condition-specific reporter signal with plasmid control, and Student's t-tests compared normalized non-reference/reference log2 ratios for enVars.

Processed data

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Column dictionary · 99 definitions

variant_id: dbSNP rs identifier for the tested variant.
tested_oligo_id: Source oligo identifier for the tested non-reference allele.
reference_oligo_id: Source oligo identifier for the reference allele paired with the tested allele.
tested_allele_label: Source allele label, such as Non-Ref or Non-Ref-1 for multiallelic variants.
reference_allele_label: Source reference allele label (Ref).
tested_allele: Nucleotide or indel allele represented by the tested oligo.
reference_allele: Reference nucleotide or indel allele represented by the reference oligo.
tested_oligo_sequence: Forward-strand synthesized oligo sequence for the tested allele; SNP records are nominally 200 bp including cloning-flank sequence, while indel records can be shorter.
reference_oligo_sequence: Forward-strand synthesized oligo sequence for the reference allele; SNP records are nominally 200 bp including cloning-flank sequence, while indel records can be shorter.
chromosome: Chromosome from Supplementary Data 2, on hg19.
position_hg19: Variant coordinate on hg19 as reported by the source.
cytoband: Cytogenetic band for the variant.
variant_annotation: dbSNP/UCSC variant-class annotation reported by the study.
tag_variant: Lead/tag SLE-risk variant defining the associated risk locus.
sle_variant: Boolean indicating whether the variant was an SLE-associated variant in the source list.
calibration_control_variant: Boolean indicating whether the variant was one of the 20 calibration controls.
nearest_gene: Nearest gene annotation used by the study.
looping_promoter_genes: Genes whose promoters were connected to the variant by GM12878 promoter-looping data.
eqtl_immune_spleen_blood: Combined eQTL genes from immune-cell, spleen, or whole-blood sources.
eqtl_lcls: Combined eQTL genes from lymphoblastoid cell-line sources.
eqtl_primary_b_cell: Combined eQTL genes from primary B-cell sources.
eqtl_b_cells_with_and_without_ebv: Combined eQTL genes from B cells with and without EBV transformation.
eqtl_primary_t_cell: Combined eQTL genes from primary T-cell sources.
eqtl_all: Combined eQTL genes across all source classes.
all_target_genes: Study-defined target-gene union from promoter looping and B-cell eQTL annotations, or nearest gene when unavailable.
gm12878_published_envar: Published Supplementary Data 2 flag for enhancer variant status in GM12878.
gm12878_published_allelic_envar: Published Supplementary Data 2 flag for allelic enhancer variant status in GM12878.
jurkat_published_allelic_envar: Published Supplementary Data 2 flag for allelic enhancer variant status in untreated Jurkat.
jurkat_tnfa_published_allelic_envar: Published Supplementary Data 2 flag for allelic enhancer variant status in TNFα-stimulated Jurkat.
gm12878_risk_locus: Cytogenetic risk-locus label from the 51-allelic-enVar annotation.
gm12878_sle_risk_allele: SLE risk allele from the GM12878 51-allelic-enVar annotation.
gm12878_risk_allele_major_minor: Whether the annotated SLE risk allele is major or minor.
gm12878_risk_allele_activity_relative_to_nonrisk: Published direction of the GM12878 risk-allele enhancer activity relative to the non-risk allele.
gm12878_risk_candidate_genes: Candidate genes listed with the GM12878 51-allelic-enVar annotation.
gm12878_risk_tier: Study annotation tier for the GM12878 allelic-enVar target-gene evidence.
gm12878_risk_tag_variant: Tag variant from the GM12878 51-allelic-enVar annotation.
gm12878_risk_ancestry: Ancestry label for the associated SLE risk signal.
unique_barcode_count_reference: Number of unique plasmid-associated barcodes (Unique_Tag) for the reference oligo.
unique_barcode_count_tested: Number of unique plasmid-associated barcodes (Unique_Tag) for the tested oligo.
plasmid_control_count_reference: Raw unique-barcode count in the plasmid control for the reference oligo.
plasmid_control_count_tested: Raw unique-barcode count in the plasmid control for the tested oligo.
reference_jurkat_dna_rep1_count: Unique-barcode count in Jurkat extracted DNA for reference allele biological replicate 1.
tested_jurkat_dna_rep1_count: Unique-barcode count in Jurkat extracted DNA for tested allele biological replicate 1.
reference_jurkat_dna_rep2_count: Unique-barcode count in Jurkat extracted DNA for reference allele biological replicate 2.
tested_jurkat_dna_rep2_count: Unique-barcode count in Jurkat extracted DNA for tested allele biological replicate 2.
reference_jurkat_dna_rep3_count: Unique-barcode count in Jurkat extracted DNA for reference allele biological replicate 3.
tested_jurkat_dna_rep3_count: Unique-barcode count in Jurkat extracted DNA for tested allele biological replicate 3.
reference_jurkat_dna_rep4_count: Unique-barcode count in Jurkat extracted DNA for reference allele biological replicate 4.
tested_jurkat_dna_rep4_count: Unique-barcode count in Jurkat extracted DNA for tested allele biological replicate 4.
reference_jurkat_dna_rep5_count: Unique-barcode count in Jurkat extracted DNA for reference allele biological replicate 5.
tested_jurkat_dna_rep5_count: Unique-barcode count in Jurkat extracted DNA for tested allele biological replicate 5.
reference_jurkat_dna_mean_count: Arithmetic mean of the five Jurkat DNA counts for the reference allele.
tested_jurkat_dna_mean_count: Arithmetic mean of the five Jurkat DNA counts for the tested allele.
reference_jurkat_rna_rep1_count: Unique-barcode count in Jurkat reporter RNA for reference allele biological replicate 1.
tested_jurkat_rna_rep1_count: Unique-barcode count in Jurkat reporter RNA for tested allele biological replicate 1.
reference_jurkat_rna_rep2_count: Unique-barcode count in Jurkat reporter RNA for reference allele biological replicate 2.
tested_jurkat_rna_rep2_count: Unique-barcode count in Jurkat reporter RNA for tested allele biological replicate 2.
reference_jurkat_rna_rep3_count: Unique-barcode count in Jurkat reporter RNA for reference allele biological replicate 3.
tested_jurkat_rna_rep3_count: Unique-barcode count in Jurkat reporter RNA for tested allele biological replicate 3.
reference_jurkat_rna_rep4_count: Unique-barcode count in Jurkat reporter RNA for reference allele biological replicate 4.
tested_jurkat_rna_rep4_count: Unique-barcode count in Jurkat reporter RNA for tested allele biological replicate 4.
reference_jurkat_rna_rep5_count: Unique-barcode count in Jurkat reporter RNA for reference allele biological replicate 5.
tested_jurkat_rna_rep5_count: Unique-barcode count in Jurkat reporter RNA for tested allele biological replicate 5.
reference_jurkat_rna_mean_count: Arithmetic mean of the five Jurkat reporter-RNA counts for the reference allele.
tested_jurkat_rna_mean_count: Arithmetic mean of the five Jurkat reporter-RNA counts for the tested allele.
reference_base_mean: DESeq2 intermediate mean of normalized counts across samples for the reference oligo.
tested_base_mean: DESeq2 intermediate mean of normalized counts across samples for the tested oligo.
reference_log2_activity: DESeq2 log2 fold change of untreated Jurkat reporter signal versus plasmid control for the reference oligo.
tested_log2_activity: DESeq2 log2 fold change of untreated Jurkat reporter signal versus plasmid control for the tested oligo.
reference_lfc_se: DESeq2 standard error of the reference-oligo log2 activity estimate.
tested_lfc_se: DESeq2 standard error of the tested-oligo log2 activity estimate.
reference_activity_stat: DESeq2 Wald statistic for reference-oligo activity versus plasmid control.
tested_activity_stat: DESeq2 Wald statistic for tested-oligo activity versus plasmid control.
reference_activity_p_value: DESeq2 Wald p-value for reference-oligo activity versus plasmid control.
tested_activity_p_value: DESeq2 Wald p-value for tested-oligo activity versus plasmid control.
reference_activity_fdr: DESeq2 Benjamini–Hochberg adjusted p-value for the reference oligo.
tested_activity_fdr: DESeq2 Benjamini–Hochberg adjusted p-value for the tested oligo.
reference_activity_fold_change: DESeq2 fold change of untreated Jurkat reporter signal versus plasmid control for the reference oligo.
tested_activity_fold_change: DESeq2 fold change of untreated Jurkat reporter signal versus plasmid control for the tested oligo.
reference_enhancer_allele: Derived Boolean: reference oligo has DESeq2 FDR < 0.05 and FoldChange >= 1.5.
tested_enhancer_allele: Derived Boolean: tested oligo has DESeq2 FDR < 0.05 and FoldChange >= 1.5.
variant_has_enhancer_allele: Derived Boolean: at least one QC-passing allele of the variant meets the enhancer-allele criteria in untreated Jurkat.
allelic_comparison: Published Student's t-test comparison key for tested non-reference allele versus reference allele.
allelic_ratio_rep1: Published non-reference/reference normalized activity ratio for allelic replicate 1.
allelic_log2_ratio_rep1: Log2 of the published non-reference/reference activity ratio for allelic replicate 1.
allelic_ratio_rep2: Published non-reference/reference normalized activity ratio for allelic replicate 2.
allelic_log2_ratio_rep2: Log2 of the published non-reference/reference activity ratio for allelic replicate 2.
allelic_ratio_rep3: Published non-reference/reference normalized activity ratio for allelic replicate 3.
allelic_log2_ratio_rep3: Log2 of the published non-reference/reference activity ratio for allelic replicate 3.
allelic_ratio_rep4: Published non-reference/reference normalized activity ratio for allelic replicate 4.
allelic_log2_ratio_rep4: Log2 of the published non-reference/reference activity ratio for allelic replicate 4.
allelic_ratio_rep5: Published non-reference/reference normalized activity ratio for allelic replicate 5.
allelic_log2_ratio_rep5: Log2 of the published non-reference/reference activity ratio for allelic replicate 5.
allelic_log2_average: Mean of the published five-replicate allelic log2 ratios.
allelic_fold_change: Published averaged fold change of tested non-reference over reference activity.
allelic_p_value: Two-sided Student's t-test p-value for the allelic comparison.
allelic_fdr: Benjamini–Hochberg FDR for the allelic comparison.
allelic_fdr_significant: Boolean indicating source Student's t-test Significance = Sig (pFDR < 0.05).
published_allelic_envar: Published Supplementary Data 2 Boolean for allelic-enVar status in untreated Jurkat.

Quality control

The authors retained oligos with at least 30 unique plasmid-associated barcodes and used five biological replicates for Jurkat. DESeq2 used Benjamini–Hochberg FDR < 0.05; an enhancer allele (enAllele) additionally required FoldChange >= 1.5. Allelic testing was limited to enVars and used two-sided Student's t-tests on normalized non-reference/reference log2 ratios with BH FDR < 0.05 and a reported >=25% fold-change requirement. Package QC retained 3,096 alternate/reference rows representing 3,043 variants: both oligos had Unique_Tag >= 30, positive plasmid counts, finite published DESeq2 statistics, and finite Jurkat DNA/RNA replicate counts; 50 of 3,146 possible alternate-allele pairs were excluded. Non-significant but QC-passing pairs remain in the table.

Curation notes

The processed table is one row per non-reference allele paired with its reference allele; multiallelic variants therefore have multiple rows. Reverse-complement synthesis records are excluded from the processed table but remain in raw Supplementary Data 4. The source t-test workbook only contains enVars, so blank allelic fields for other QC-passing variants indicate that no allelic test was reported, not that the MPRA measurement was absent. The Jurkat cell line is resolved to the parent/standard clone Cellosaurus CVCL:0367 (Jurkat E6.1).

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