Experiment / E5XEUD6L9Other

Nonintegrating 5'/5' MT lentiMPRA of HepG2 candidate liver enhancers

A systematic evaluation of the design and context dependencies of massively parallel reporter assays

The common 2,440-element library was packaged with mutant lentiviral integrase, leaving the 171-bp enhancer/minimal-promoter reporter library nonintegrated/episomal with linked barcodes in the reporter 5' UTR. Three independent HepG2 infections were profiled by matched DNA and RNA barcode sequencing.

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Organism: Human
Taxonomy ID: NCBITaxon:9606
Biosample: CVCL:0027
Reference genome: hg19
Design focus: Region-focused
Region of interest: Not reported / not applicable

Perturbation & assay details

Basal / Untreated

The 5'/5' MT design matches the 5'/5' WT enhancer/barcode geometry but uses mutant integrase to preserve an episomal reporter context; three independent HepG2 infections were measured.

Nonintegrating lentiviral lentiMPRA: mutant integrase kept the 5'/5' enhancer-reporter library nonintegrated/episomal; enhancer-linked RNA barcode abundance was normalized to matched DNA barcode abundance.

Processed data

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Column dictionary · 20 definitions

element_id: Published identifier for the tested 171-bp enhancer or control element.
element_type: Source element class: test, pos control, or neg control.
chromosome: Human hg19 chromosome for the source element; blank for synthetic controls without a genomic coordinate.
start: 1-based hg19 start coordinate of the 171-bp source element; blank when unavailable.
end: 1-based hg19 end coordinate of the 171-bp source element; blank when unavailable.
sequence: The 171-bp DNA sequence tested in the assay.
sequence_length_bp: Length of sequence in base pairs.
activity_mean_log2_rna_dna: Authors' activity score: mean log2 RNA/DNA after per-replicate median normalization; the source excludes the low-quality replicate 1 for 5'/3' MT and 3'/3' MT.
replicate_1_normalized_rna_cpm: Normalized RNA barcode count for replicate 1, reported as counts per million in the authors' pipeline.
replicate_1_normalized_dna_cpm: Normalized DNA barcode count for replicate 1, reported as counts per million in the authors' pipeline.
replicate_1_log2_rna_dna_ratio: Raw per-replicate log2(RNA/DNA) activity value for replicate 1, before the across-replicate median normalization used for the published mean.
replicate_1_unique_barcodes: Number of unique barcodes assigned to the element in replicate 1.
replicate_2_normalized_rna_cpm: Normalized RNA barcode count for replicate 2, reported as counts per million in the authors' pipeline.
replicate_2_normalized_dna_cpm: Normalized DNA barcode count for replicate 2, reported as counts per million in the authors' pipeline.
replicate_2_log2_rna_dna_ratio: Raw per-replicate log2(RNA/DNA) activity value for replicate 2, before the across-replicate median normalization used for the published mean.
replicate_2_unique_barcodes: Number of unique barcodes assigned to the element in replicate 2.
replicate_3_normalized_rna_cpm: Normalized RNA barcode count for replicate 3, reported as counts per million in the authors' pipeline.
replicate_3_normalized_dna_cpm: Normalized DNA barcode count for replicate 3, reported as counts per million in the authors' pipeline.
replicate_3_log2_rna_dna_ratio: Raw per-replicate log2(RNA/DNA) activity value for replicate 3, before the across-replicate median normalization used for the published mean.
replicate_3_unique_barcodes: Number of unique barcodes assigned to the element in replicate 3.

Quality control

Author QC was retained: barcode-to-element associations required at least three independent observations and at least 90% of barcode sequence mapped to one element; only barcodes observed in both the DNA and RNA sample were counted; UMI-collapsed counts were normalized to counts per million; and replicate RNA/DNA ratios were median-normalized before averaging. Package QC retained only rows with a valid A/C/G/T 171-bp sequence, finite published mean and replicate activity values, and finite source RNA, DNA, and unique-barcode fields for all three replicates. 2429 rows passed from 2429 source mean-score rows; 0 incomplete/missing rows were excluded. No extra minimum-barcode cutoff was imposed because the paper uses its measured element set for assay comparisons; observed barcode counts remain in the table.

Curation notes

All 2429 complete elements in the authors' 2429-row all-assay mean table were retained; no replicate was excluded from this assay's published mean.

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