Experiment / E3NS1W4D7Episomal Plasmid MPRA

HepG2 MPRA — tunicamycin-induced ER stress

Genome-wide census of ATF4 binding sites and functional profiling of trait-associated genetic variants overlapping ATF4 binding motifs

An episomal barcode MPRA tested reference and alternative alleles of 581 trait-associated SNP-containing 175-bp sequences in HepG2 cells after tunicamycin-induced endoplasmic-reticulum stress. Five independent transfections performed on separate days were analyzed by poly(A)+ RNA barcode sequencing and MPRAnalyze.

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Organism: Human
Taxonomy ID: NCBITaxon:9606
Biosample: CVCL:0027
Reference genome: GRCh38
Design focus: Variant-focused
Region of interest: Not reported / not applicable

Perturbation & assay details

2 µg/ml tunicamycin for 17 h after 9 h post-transfection recovery

The pMPRA1/pMPRAdonor2 plasmid configuration placed each 175-nt query sequence (SNP centered with 87-bp flanks) upstream of a minimal promoter and luc2 ORF, with a unique 10-nt barcode in the reporter transcript. Each allele was synthesized with six barcodes; 6×10^6 HepG2 cells per replicate were transfected with 22 µg library using Lipofectamine 3000. Nine hours after transfection the medium was replaced with regular medium containing 2 µg/ml tunicamycin, and cells were harvested 17 h later. Poly(A)+ RNA and plasmid-input DNA were sequenced on an Illumina NextSeq 500; the RNA read used a 12-nt UMI for PCR-duplicate correction.

Processed data

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Column dictionary · 44 definitions

element_id: Package identifier for the tested SNP element.
variant_id: dbSNP rsID of the assayed variant.
variant_key: Author-provided GRCh38 variant and cloning-strand label.
chromosome: GRCh38 chromosome name.
position_grch38: 1-based GRCh38 SNP coordinate.
ref_allele: Reference allele in the GRCh38 variant label.
alt_allele: Alternative allele in the GRCh38 variant label.
cloned_strand: Sequence orientation used for cloning: positive or negative genomic strand.
ref_query_sequence_175bp: 175-bp reference-allele query sequence used in the MPRA.
alt_query_sequence_175bp: 175-bp alternative-allele query sequence used in the MPRA.
ref_qc_barcode_count: Number of reference-allele barcodes passing the published source-QC filter.
alt_qc_barcode_count: Number of alternative-allele barcodes passing the published source-QC filter.
ref_qc_barcodes: Pipe-delimited 10-nt reference barcode sequences retained by source QC.
alt_qc_barcodes: Pipe-delimited 10-nt alternative barcode sequences retained by source QC.
ref_input_count_mean: Mean raw DNA-input count across retained reference barcodes and four input libraries.
alt_input_count_mean: Mean raw DNA-input count across retained alternative barcodes and four input libraries.
ref_rna_count_mean: Mean raw RNA count across retained reference barcodes and the five tunicamycin RNA libraries.
alt_rna_count_mean: Mean raw RNA count across retained alternative barcodes and the five tunicamycin RNA libraries.
ref_activity_rep1: Author-normalized reference reporter activity for tunicamycin independent replicate 1.
ref_activity_rep2: Author-normalized reference reporter activity for tunicamycin independent replicate 2.
ref_activity_rep3: Author-normalized reference reporter activity for tunicamycin independent replicate 3.
ref_activity_rep4: Author-normalized reference reporter activity for tunicamycin independent replicate 4.
ref_activity_rep5: Author-normalized reference reporter activity for tunicamycin independent replicate 5.
alt_activity_rep1: Author-normalized alternative reporter activity for tunicamycin independent replicate 1.
alt_activity_rep2: Author-normalized alternative reporter activity for tunicamycin independent replicate 2.
alt_activity_rep3: Author-normalized alternative reporter activity for tunicamycin independent replicate 3.
alt_activity_rep4: Author-normalized alternative reporter activity for tunicamycin independent replicate 4.
alt_activity_rep5: Author-normalized alternative reporter activity for tunicamycin independent replicate 5.
ref_activity_mean: Mean of the five tunicamycin reference reporter-activity values.
alt_activity_mean: Mean of the five tunicamycin alternative reporter-activity values.
mpranalyze_ref_alt_log2fc: MPRAnalyze REF/ALT allelic log2 fold change; positive values indicate greater reference activity.
mpranalyze_model_logfc: MPRAnalyze model log fold-change estimate for the allelic comparison.
mpranalyze_statistic: MPRAnalyze allelic-effect test statistic.
mpranalyze_pval: Unadjusted MPRAnalyze allelic-effect p-value.
mpranalyze_fdr: Benjamini-Hochberg FDR for the MPRAnalyze allelic-effect test.
mpra_allelic_effect_significant: Author’s yes/no flag for MPRAnalyze FDR < 0.05 in tunicamycin-treated cells.
higher_activity_allele: Allele with higher activity without a p-value cutoff, as reported by the authors.
motif_consequence: Author-predicted ATF4-associated motif consequence of the alternative allele.
motif_directionally_concordant: Author’s no-p-value-cutoff indicator that MPRA direction agrees with motif prediction.
motif_and_mpra_direction_concordance_fdr05: Author’s motif/MPRA direction-concordance result after the FDR < 0.05 cutoff.
genomic_location: Author annotation as promoter or distal; promoter is TSS ±1 kb.
tss_strand: Strand of the nearby TSS reported by the authors, when present.
tss_gene: Nearby TSS gene reported by the authors, when present.
tss_distance_bp: Distance in bp to the reported nearby TSS, when present.

Quality control

The authors trimmed reads to 35 bp, mapped to barcode-plus-constant synthetic contigs with Bowtie allowing one mismatch, discarded multimappers, deduplicated UMIs with UMI-tools, and normalized counts to sequencing depth, DNA input, and median scramble-control activity. Barcodes were retained when they had ≥5 reads in every one of four DNA-input replicates and, in at least one treatment, ≥5 reads in every one of five RNA replicates; MPRAnalyze FDR < 0.05 defined a significant allelic effect. For this package, the same source-QC filter was applied to the GEO matrix and both REF and ALT constructs were required to have at least one retained barcode; all 581 variant rows passed. The downloaded matrix yields 6,985 retained barcodes, while the article’s processing paragraph reports 6,983 and its S7 caption reports 6,985.

Curation notes

The table is variant-level rather than a control library table and combines S13 construct sequences, S14 normalized activity, S15 MPRAnalyze results, and GEO barcode counts. The source filter is condition-agnostic (a barcode passes if it meets the RNA threshold in either condition), matching the paper’s published processing; all 581 variants retain both alleles in this experiment. HepG2 is represented by Cellosaurus CVCL:0027 (Hep-G2).

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