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Nonintegrating 5'/3' 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 171-bp candidate enhancers upstream of a minimal promoter with linked barcodes in the reporter 3' UTR as nonintegrated/episomal reporter DNA. 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'/3' MT construct uses the same enhancer/barcode geometry as 5'/3' WT, but mutant integrase prevents genomic integration; three independent HepG2 infections were measured.

Nonintegrating lentiviral lentiMPRA: mutant lentiviral integrase was used to keep the pLS-mP enhancer-reporter library nonintegrated/episomal; enhancer-linked RNA barcode abundance was normalized to matched DNA barcode abundance.

Processed data

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Filters apply to this table only. The CSV download contains the complete processed table; filtered rows are available through the API.

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. 2425 rows passed from 2429 source mean-score rows; 4 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

The table retains 2425 complete elements from the authors' 2429-row all-assay mean table; 4 rows with missing or incomplete assay-specific source fields were omitted. The authors excluded replicate 1 from the published mean for this mutant-integrase assay because of low barcode count and poor sample quality; its raw per-replicate fields remain in the table for transparency.

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