Experiment / E11C52A3DEpisomal Plasmid MPRA

TNFα-stimulated 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, followed by TNFα stimulation. Matched DNA and reporter RNA barcode counts were used to quantify enhancer activity and published allelic Student's t-test results were retained 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

100 ng/mL TNFα for 24 h after 24 h post-electroporation recovery

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, supplemented with 100 ng/mL TNFα for a further 24 h, and collected for matched DNA and RNA barcode sequencing. DESeq2 compared TNFα-condition 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_tnfa_dna_rep1_count: Unique-barcode count in TNFα-treated Jurkat extracted DNA for reference allele biological replicate 1.
tested_jurkat_tnfa_dna_rep1_count: Unique-barcode count in TNFα-treated Jurkat extracted DNA for tested allele biological replicate 1.
reference_jurkat_tnfa_dna_rep2_count: Unique-barcode count in TNFα-treated Jurkat extracted DNA for reference allele biological replicate 2.
tested_jurkat_tnfa_dna_rep2_count: Unique-barcode count in TNFα-treated Jurkat extracted DNA for tested allele biological replicate 2.
reference_jurkat_tnfa_dna_rep3_count: Unique-barcode count in TNFα-treated Jurkat extracted DNA for reference allele biological replicate 3.
tested_jurkat_tnfa_dna_rep3_count: Unique-barcode count in TNFα-treated Jurkat extracted DNA for tested allele biological replicate 3.
reference_jurkat_tnfa_dna_rep4_count: Unique-barcode count in TNFα-treated Jurkat extracted DNA for reference allele biological replicate 4.
tested_jurkat_tnfa_dna_rep4_count: Unique-barcode count in TNFα-treated Jurkat extracted DNA for tested allele biological replicate 4.
reference_jurkat_tnfa_dna_rep5_count: Unique-barcode count in TNFα-treated Jurkat extracted DNA for reference allele biological replicate 5.
tested_jurkat_tnfa_dna_rep5_count: Unique-barcode count in TNFα-treated Jurkat extracted DNA for tested allele biological replicate 5.
reference_jurkat_tnfa_dna_mean_count: Arithmetic mean of the five TNFα-treated Jurkat DNA counts for the reference allele.
tested_jurkat_tnfa_dna_mean_count: Arithmetic mean of the five TNFα-treated Jurkat DNA counts for the tested allele.
reference_jurkat_tnfa_rna_rep1_count: Unique-barcode count in TNFα-treated Jurkat reporter RNA for reference allele biological replicate 1.
tested_jurkat_tnfa_rna_rep1_count: Unique-barcode count in TNFα-treated Jurkat reporter RNA for tested allele biological replicate 1.
reference_jurkat_tnfa_rna_rep2_count: Unique-barcode count in TNFα-treated Jurkat reporter RNA for reference allele biological replicate 2.
tested_jurkat_tnfa_rna_rep2_count: Unique-barcode count in TNFα-treated Jurkat reporter RNA for tested allele biological replicate 2.
reference_jurkat_tnfa_rna_rep3_count: Unique-barcode count in TNFα-treated Jurkat reporter RNA for reference allele biological replicate 3.
tested_jurkat_tnfa_rna_rep3_count: Unique-barcode count in TNFα-treated Jurkat reporter RNA for tested allele biological replicate 3.
reference_jurkat_tnfa_rna_rep4_count: Unique-barcode count in TNFα-treated Jurkat reporter RNA for reference allele biological replicate 4.
tested_jurkat_tnfa_rna_rep4_count: Unique-barcode count in TNFα-treated Jurkat reporter RNA for tested allele biological replicate 4.
reference_jurkat_tnfa_rna_rep5_count: Unique-barcode count in TNFα-treated Jurkat reporter RNA for reference allele biological replicate 5.
tested_jurkat_tnfa_rna_rep5_count: Unique-barcode count in TNFα-treated Jurkat reporter RNA for tested allele biological replicate 5.
reference_jurkat_tnfa_rna_mean_count: Arithmetic mean of the five TNFα-treated Jurkat reporter-RNA counts for the reference allele.
tested_jurkat_tnfa_rna_mean_count: Arithmetic mean of the five TNFα-treated 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 TNFα-treated Jurkat reporter signal versus plasmid control for the reference oligo.
tested_log2_activity: DESeq2 log2 fold change of TNFα-treated 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 TNFα-treated Jurkat reporter signal versus plasmid control for the reference oligo.
tested_activity_fold_change: DESeq2 fold change of TNFα-treated 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 TNFα-treated 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 TNFα-stimulated Jurkat.

Quality control

The authors retained oligos with at least 30 unique plasmid-associated barcodes and used five biological replicates for TNFα-treated 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 2,929 alternate/reference rows representing 2,879 variants: both oligos had Unique_Tag >= 30, positive plasmid counts, finite published TNFα DESeq2 statistics, and finite TNFα DNA/RNA replicate counts; 217 of 3,146 possible alternate-allele pairs were excluded, including source rows with non-finite/NA TNFα DESeq2 results. 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 TNFα DESeq2 sheet contains 240 rows with NA statistics; those and any pair member failing the stated barcode/result QC were excluded. 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.

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