Experiment / E53CCZ9ODIntegrated lentiMPRA

Three-replicate integrated lentiMPRA in induced human excitatory neurons

Massively parallel reporter assays and mouse transgenic assays provide correlated and complementary information about neuronal enhancer activity

A 270-bp lentiMPRA library containing neuronal regulatory-element tiles, scrambled negative controls, and single-base or multi-variant constructs was integrated into WTC11-Ngn2 induced human excitatory neurons. DNA and transcribed RNA barcodes were measured in three biological replicates to quantify element activity and variant-versus-reference effects.

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Organism: Human
Taxonomy ID: NCBITaxon:9606
Biosample: CL:0000679
Reference genome: GRCh38
Design focus: Variant-focused
Region of interest: Not reported / not applicable

Perturbation & assay details

Basal / Untreated

Oligos were cloned upstream of a minimal promoter in a barcoded lentiMPRA vector and delivered by lentivirus at MOI 80 to differentiated WTC11-Ngn2 iPSC-derived human excitatory neurons. Each insert received a random 15-bp barcode; integrated DNA and barcode-derived RNA were sequenced separately for three biological replicates. MPRAflow-derived activity is the log2 RNA/DNA ratio, with the combined public activity table reporting the mean across replicates and a z-score relative to scrambled negative controls. The mixed library includes GRCh38 genomic tiles from neuronal ATAC-seq/VISTA enhancer inputs, 24,942 designed variant constructs, and 500 designed scrambled negative controls.

Processed data

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Column dictionary · 44 definitions

construct_id: Original ENCODE construct name combining element identifier, coordinate label, and construct suffix.
base_element_id: Library tile or element identifier before the coordinate and construct suffix.
coordinate_label: Source coordinate label containing chromosome, 1-based start/end, and strand sign.
chromosome: Chromosome parsed from coordinate_label.
genomic_start_1based: 1-based inclusive genomic start parsed from coordinate_label.
genomic_end_1based: 1-based inclusive genomic end parsed from coordinate_label.
strand: Source sequence orientation sign, either + or -.
bed_start_0based: 0-based half-open BED start corresponding to the parsed interval.
bed_end_0based_exclusive: 0-based half-open BED end corresponding to the parsed interval.
construct_class: Package classification as a reference genomic element, scrambled negative control, single-nucleotide variant construct, or multi-SNV/VISTA alternate construct.
source_qc_type: Per-construct QC type from the authors' final_tests.rds annotation; only reference, mutation, and scramble_negative records are retained.
is_reference: Boolean indicating the reference construct suffix.
is_variant: Boolean indicating a non-reference, non-scrambled alternate construct.
is_scrambled_negative_control: Boolean indicating a scrambled negative-control construct.
variant_ref_allele: Reference allele encoded in a single-base construct suffix; blank when not applicable or unavailable.
variant_alt_allele: Alternate allele encoded in a single-base construct suffix; blank when not applicable or unavailable.
variant_position_in_tile_1based: 1-based position within the 270-bp construct encoded in a single-base variant suffix.
variant_genomic_position_1based: 1-based genomic position calculated from the tile coordinate, strand, and encoded within-tile position.
reference_construct_id: Construct ID of the paired reference tile used for variant comparisons; blank for reference and negative-control rows.
sequence: 270-bp tested construct sequence from the ENCODE design FASTA.
reference_sequence: 270-bp sequence of the paired reference construct for variant rows; blank otherwise.
rep1_rna_normalized: Normalized RNA abundance reported for biological replicate 1.
rep1_dna_normalized: Normalized integrated-library DNA abundance reported for biological replicate 1.
rep1_log2_rna_dna_ratio: Biological replicate 1 log2 RNA/DNA activity score.
rep1_barcode_count: Number of barcode tags contributing to biological replicate 1 activity.
rep2_rna_normalized: Normalized RNA abundance reported for biological replicate 2.
rep2_dna_normalized: Normalized integrated-library DNA abundance reported for biological replicate 2.
rep2_log2_rna_dna_ratio: Biological replicate 2 log2 RNA/DNA activity score.
rep2_barcode_count: Number of barcode tags contributing to biological replicate 2 activity.
rep3_rna_normalized: Normalized RNA abundance reported for biological replicate 3.
rep3_dna_normalized: Normalized integrated-library DNA abundance reported for biological replicate 3.
rep3_log2_rna_dna_ratio: Biological replicate 3 log2 RNA/DNA activity score.
rep3_barcode_count: Number of barcode tags contributing to biological replicate 3 activity.
mean_log2_rna_dna_ratio: Mean of the three replicate log2 RNA/DNA ratios; equal to the deposited combined activity ratio.
replicate_sd_log2_rna_dna_ratio: Sample standard deviation of the three replicate log2 RNA/DNA ratios.
min_barcode_count: Minimum barcode tag count across the three biological replicates.
activity_zscore_negative_control: Deposited combined activity z-score relative to scrambled negative controls.
reference_activity_log2_rna_dna_ratio: Combined log2 RNA/DNA activity ratio of the paired reference construct; blank when not applicable.
reference_activity_zscore_negative_control: Combined negative-control z-score of the paired reference construct; blank when not applicable.
variant_delta_log2_rna_dna_ratio: Variant or alternate construct activity ratio minus paired reference activity ratio.
variant_delta_activity_zscore: Variant or alternate construct negative-control z-score minus paired reference z-score.
variant_effect_direction: Unthresholded direction of the variant-minus-reference z-score difference; not a statistical significance call.
selected_for_mouse_transgenic_followup: Boolean indicating one of the five constructs listed in the study source tested_variants.tsv for mouse transgenic follow-up.
mouse_transgenic_vista_id: VISTA enhancer identifier associated with a selected mouse transgenic follow-up construct; blank otherwise.

Quality control

The paper retained tiles with at least 15 detected barcodes in each of three biological replicates, discarded mutation tiles lacking a qualifying reference tile, and excluded barcodes whose normalized log2 RNA/DNA standard deviation across replicates exceeded 1. Reported replicate Pearson correlations were 0.76–0.78 and the paper reports 73,367 of 81,952 designed elements passing its final QC. The authors' final_tests.rds annotation labels records with source QC types reference, mutation, scramble_negative, not_enough_barcodes, or orphaned. Package-level QC retained combined-table records with an allowed source QC type, numeric activity values, a matching record in all three replicate activity files, at least 15 barcode tags in every replicate, a 270-bp sequence in the deposited design FASTA, and a paired reference construct for every variant-like record. Of 76,415 records in the deposited combined ENCODE activity table, 73,187 passed these checks and were retained: 50,083 reference elements, 22,530 single-nucleotide variant constructs, 120 multi-SNV/VISTA alternate constructs, and 454 scrambled negative controls. The excluded public records were 3,111 source-not_enough_barcodes records and 117 source-orphaned records.

Curation notes

This is one MPRA experiment with three biological replicates; the mouse enSERT/H11 lacZ assays are orthogonal validation and are not packaged as a second MPRA experiment. CL:0000679 (glutamatergic neuron) is used as the terminal Cell Ontology term for the described induced excitatory neurons. The library is mixed, but Variant-focused is used because allelic and saturation-mutagenesis effects are a central study objective; the table retains both reference enhancer tiles and variant constructs. The public combined ENCODE activity.tsv contains 76,415 construct records. The compact source final_tests.rds has a per-construct type annotation that yields 73,187 passing public records when reference, mutation, and scramble_negative are accepted; this differs slightly from the article narrative count of 73,367, so the source RDS is retained in raw_data and the applied source-type filter is recorded explicitly. The RDS keep field is an element-level flag for reference/scramble analysis rather than the complete variant QC set. Blank variant/reference fields indicate not applicable or unavailable values; the effect-direction columns are descriptive activity differences without published adjusted p-values in the deposited activity TSV.

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