Experiment / E0EIQQASHOther

Nonintegrating lentiMPRA of HepG2 candidate liver enhancers (mutant integrase / MT)

A systematic comparison reveals substantial differences in chromosomal versus episomal encoding of enhancer activity

The 2,440-element 5′ enhancer/3′ barcode library was packaged with a mutant lentiviral integrase and introduced into HepG2 cells, leaving the reporter library nonintegrated/episomal. Three independent infections were profiled by matched DNA and mRNA barcode sequencing for 2,236 candidate liver enhancers and 204 controls.

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Organism: Human
Taxonomy ID: NCBITaxon:9606
Biosample: CVCL:0027
Reference genome: GRCh37
Design focus: Region-focused
Region of interest: Not reported / not applicable

Perturbation & assay details

Basal / untreated; mutant-integrase lentiviral delivery

The library used a 5′ 171-bp candidate sequence upstream of a minimal promoter and EGFP, with a 15-bp barcode in the EGFP 3′ UTR; each insert had 100 designed barcodes and the cassette was flanked by antirepressor/SAR elements. HepG2 DNA and mRNA were collected at day 3 after an estimated 100 viral particles per cell; three independent infections were sequenced on Illumina NextSeq.

Nonintegrating lentiviral lentiMPRA: a mutant lentiviral integrase kept a pLS-mP enhancer-EGFP reporter library as nonintegrated/episomal DNA. Reporter RNA barcode abundance was normalized to matched DNA barcode abundance to quantify enhancer activity.

Processed data

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Column dictionary · 53 definitions

element_id: Published insert/region identifier from GEO ActivityRatios.
design_region_id: Identifier in the oligo design; differs from element_id only for four controls whose activity labels add a bracketed coordinate span.
category: Broad published design category: type1–type4, SRE, or control.
category_detail: Published subtype or control label, such as HNF4A-ChMod, positive, or negative.
element_sequence: The intended 171-bp enhancer or control sequence extracted from the designed 230-bp oligo.
sequence_length_bp: Length of element_sequence in base pairs.
coordinate_assembly: Assembly label parsed from the published identifier; candidate regions use GRCh37 and controls preserve hg18, hg19, or mm9 labels.
coordinate: High-level target interval parsed from the published region identifier.
chromosome: Chromosome parsed from coordinate.
start: 1-based target interval start parsed from coordinate.
end: Target interval end parsed from coordinate.
barcode_count_designed: Number of designed 15-bp reporter barcodes assigned to the insert; the library design used 100.
activity_rep1_rna_dna: Published mutant-integrase/nonintegrating RNA/DNA activity ratio for replicate 1.
activity_rep2_rna_dna: Published mutant-integrase/nonintegrating RNA/DNA activity ratio for replicate 2.
activity_rep3_rna_dna: Published mutant-integrase/nonintegrating RNA/DNA activity ratio for replicate 3.
activity_combined_rna_dna: Published MT activity ratio combined across replicates after replicate-median normalization.
activity_log2_rna_dna: log2 of activity_combined_rna_dna, derived in this package.
replicate_activity_mean_rna_dna: Arithmetic mean of the three published MT replicate ratios; descriptive and not the authors’ combined value.
replicate_activity_sd_rna_dna: Sample standard deviation of the three published MT replicate ratios.
barcodes_common_rep1: Designed barcode IDs observed in both DNA and RNA count files for MT replicate 1.
barcodes_common_rep2: Designed barcode IDs observed in both DNA and RNA count files for MT replicate 2.
barcodes_common_rep3: Designed barcode IDs observed in both DNA and RNA count files for MT replicate 3.
barcodes_common_mean: Arithmetic mean of the three shared RNA/DNA barcode counts.
dna_counts_common_rep1: Sum of assigned DNA counts over barcodes shared by RNA and DNA in MT replicate 1.
dna_counts_common_rep2: Sum of assigned DNA counts over barcodes shared by RNA and DNA in MT replicate 2.
dna_counts_common_rep3: Sum of assigned DNA counts over barcodes shared by RNA and DNA in MT replicate 3.
rna_counts_common_rep1: Sum of assigned RNA counts over barcodes shared by RNA and DNA in MT replicate 1.
rna_counts_common_rep2: Sum of assigned RNA counts over barcodes shared by RNA and DNA in MT replicate 2.
rna_counts_common_rep3: Sum of assigned RNA counts over barcodes shared by RNA and DNA in MT replicate 3.
paired_other_context_activity_combined_rna_dna: Published combined WT/integrated RNA/DNA activity ratio for the paired context.
integrated_minus_episomal_log2_ratio: log2(WT combined activity / MT combined activity); positive values favor the integrated context.
qc_pass: TRUE for rows retained after package-level design, activity, and shared-barcode QC.
OC: Majority HepG2 OpenChromatin state annotation.
SegWay: Majority HepG2 SegWay genome-segmentation annotation.
ChromHMM7: Majority HepG2 7-state ChromHMM annotation.
ChromHMM15: Majority Roadmap HepG2 15-state ChromHMM annotation.
ERBFeature: Majority Ensembl Regulatory Build regulatory-feature annotation.
DHS: Number of element bases overlapping HepG2 DNase hypersensitivity calls.
H3K27ac: Number of element bases covered by HepG2 H3K27ac ChIP-seq peaks.
H3K27acAve: Average HepG2 H3K27ac peak signal over the element.
H3K27acMax: Maximum HepG2 H3K27ac peak signal over the element.
TFcount: Number of distinct assayed transcription factors with ChIP-seq peak overlap.
EP300: Number of element bases covered by HepG2 EP300 ChIP-seq peaks.
FOXA1: Number of element bases covered by HepG2 FOXA1 ChIP-seq peaks.
FOXA2: Number of element bases covered by HepG2 FOXA2 ChIP-seq peaks.
HNF4A: Number of element bases covered by HepG2 HNF4A ChIP-seq peaks.
RAD21: Number of element bases covered by HepG2 RAD21 ChIP-seq peaks.
CHD2: Number of element bases covered by HepG2 CHD2 ChIP-seq peaks.
SMC3: Number of element bases covered by HepG2 SMC3 ChIP-seq peaks.
GC: CADD v1.3 average local GC percentage annotation.
CpG: CADD v1.3 average local CpG percentage annotation.
minDistTSS: CADD v1.3 distance to the closest transcribed-sequence start site.
CADD: CADD v1.3 PHRED-scaled sequence annotation.

Quality control

Author QC was retained: 15-bp barcode consensus sequences were matched to the designed barcode set without clustering; only barcodes observed in both RNA and DNA in the same sample were used; counts were normalized to counts per million; per-insert RNA/DNA ratios were calculated from summed barcode counts; replicate ratios were divided by their replicate median before averaging. Package-level integrity QC additionally required an exact design match, 100 designed barcodes, positive numeric activity in all three replicates, and at least 3 shared RNA/DNA barcodes per replicate. All 2,440/2,440 inserts passed; the minimum shared-barcode count was 9, 8, and 9 across the three MT replicates.

Curation notes

This is the mutant-integrase arm of a paired WT/MT lentiMPRA, not a conventional transfected plasmid MPRA; it is classified as Other because the controlled assay list has no nonintegrating lentiviral option. The study is region-focused rather than variant-focused: it tests annotated 171-bp candidate liver enhancers and controls, with no designed allele contrasts. ActivityRatios contains all 2,440 inserts; source values marked NA in selected annotation fields are emitted as blank cells, while the primary activity fields are complete. The four endogenous/SRE controls include hg19, hg18, or mm9 labels in their identifiers and are retained as controls.

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