Seven-time-point perturbation lentiMPRA during human ESC neural differentiation
Massively parallel reporter perturbation assays uncover temporal regulatory architecture during neural differentiationA 10,041-sequence integrated lentiMPRA library containing wild-type, scrambled/random-control, and single- or double-motif perturbation constructs was assayed at 0, 3, 6, 12, 24, 48, and 72 hours after dual-SMAD neural induction of H1 human embryonic stem cells. This package table contains the 598 consensus functional regulatory sites (FRSs) retained by the authors' cross-method perturbation QC.
Processed tables are specific to each experiment. Column names, units, measurements, and table structure are not standardized across the database. Check this experiment’s column definitions and quality-control notes before comparing or combining data.
Perturbation & assay details
Dual-SMAD inhibition to induce neural differentiation; samples collected at 0, 3, 6, 12, 24, 48, and 72 hours after induction
The library contained 591 endogenous 171-bp human regulatory regions and 2,144 selected motif instances, with three motif-perturbation designs plus selected double perturbations, 591 WT controls, 591 SCRAM controls, and RAND controls. Constructs were cloned upstream of a minimal promoter in a pLS-SceI-derived lentiMPRA vector with 15-bp random 5-prime UTR EGFP barcodes, packaged as lentivirus, and infected into H1/WA-01 hESCs at MOI 5–8. Three independent replicate cultures were used; two received one lentivirus batch and one received a second batch. Integrated DNA barcodes and transcribed RNA barcodes were quantified at seven time points, and MPRAflow/MPRAnalyze were used to assign barcodes, estimate alpha activity, and test WT versus PERT effects.
Processed data
50 rows per page. Click a cell to inspect its full value.
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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 · 53 definitions
- frs_id
- Stable package identifier for the consensus functional regulatory site row.
- motif_id
- Motif/PWM identifier for the perturbed binding site.
- transcription_factor
- TF token parsed from the motif identifier before the first underscore.
- region
- The 171-bp endogenous regulatory region, reported as chromosome:start-end in hg19 coordinates.
- chromosome
- Chromosome parsed from region.
- region_start_hg19
- Reported hg19 region start coordinate.
- region_end_hg19
- Reported hg19 region end coordinate.
- motif_effect_code
- Source effect code: 1 essential, 2 contributing, 3 silencing, 4 inhibiting.
- motif_effect_category
- Published FRS category based on the effect of motif perturbation.
- effect_direction
- Direction of the perturbation contrast: activating motifs have positive WT-over-PERT effects; dampening motifs have negative WT-over-PERT effects.
- motif_start_1based
- 1-based start position of the motif within the tested 171-bp sequence.
- motif_end_1based
- 1-based end position of the motif within the tested 171-bp sequence.
- strand
- Motif strand in the tested sequence (+ or -).
- replaced_sequence
- Replacement sequence used to perturb the motif.
- closest_genes
- Semicolon-separated closest genes within 1 Mb, normalized from the source comma-delimited field.
- temporal_cluster_wt
- Source temporal cluster label (1–4) for the WT region.
- temporal_cluster_log2fc
- Source temporal cluster label (1–4) for the log2 fold-change trajectory.
- temporal_cluster_wt_alpha
- Source temporal cluster label (1–4) for the WT alpha trajectory.
- pchiC_hESC_genes
- Semicolon-separated PCHi-C-linked genes in hESCs, where available.
- pchiC_NPC_genes
- Semicolon-separated PCHi-C-linked genes in neural progenitor cells, where available.
- pchiC_excitatory_genes
- Semicolon-separated PCHi-C-linked genes in excitatory neurons, where available.
- pchiC_hippocampal_genes
- Semicolon-separated PCHi-C-linked genes in hippocampal cells, where available.
- pchiC_motor_genes
- Semicolon-separated PCHi-C-linked genes in motor neurons, where available.
- pchiC_astrocytes_genes
- Semicolon-separated PCHi-C-linked genes in astrocytes, where available.
- log2_wt_over_perturbed_0h
- Published MPRAnalyze log2 fold-change for WT activity divided by perturbed activity at 0 hours.
- log2_wt_over_perturbed_3h
- Published MPRAnalyze log2 fold-change for WT activity divided by perturbed activity at 3 hours.
- log2_wt_over_perturbed_6h
- Published MPRAnalyze log2 fold-change for WT activity divided by perturbed activity at 6 hours.
- log2_wt_over_perturbed_12h
- Published MPRAnalyze log2 fold-change for WT activity divided by perturbed activity at 12 hours.
- log2_wt_over_perturbed_24h
- Published MPRAnalyze log2 fold-change for WT activity divided by perturbed activity at 24 hours.
- log2_wt_over_perturbed_48h
- Published MPRAnalyze log2 fold-change for WT activity divided by perturbed activity at 48 hours.
- log2_wt_over_perturbed_72h
- Published MPRAnalyze log2 fold-change for WT activity divided by perturbed activity at 72 hours.
- wt_activity_alpha_0h
- MPRAnalyze WT sequence activity alpha at 0 hours.
- wt_activity_alpha_3h
- MPRAnalyze WT sequence activity alpha at 3 hours.
- wt_activity_alpha_6h
- MPRAnalyze WT sequence activity alpha at 6 hours.
- wt_activity_alpha_12h
- MPRAnalyze WT sequence activity alpha at 12 hours.
- wt_activity_alpha_24h
- MPRAnalyze WT sequence activity alpha at 24 hours.
- wt_activity_alpha_48h
- MPRAnalyze WT sequence activity alpha at 48 hours.
- wt_activity_alpha_72h
- MPRAnalyze WT sequence activity alpha at 72 hours.
- perturbed_activity_alpha_0h
- MPRAnalyze motif-perturbed sequence activity alpha at 0 hours.
- perturbed_activity_alpha_3h
- MPRAnalyze motif-perturbed sequence activity alpha at 3 hours.
- perturbed_activity_alpha_6h
- MPRAnalyze motif-perturbed sequence activity alpha at 6 hours.
- perturbed_activity_alpha_12h
- MPRAnalyze motif-perturbed sequence activity alpha at 12 hours.
- perturbed_activity_alpha_24h
- MPRAnalyze motif-perturbed sequence activity alpha at 24 hours.
- perturbed_activity_alpha_48h
- MPRAnalyze motif-perturbed sequence activity alpha at 48 hours.
- perturbed_activity_alpha_72h
- MPRAnalyze motif-perturbed sequence activity alpha at 72 hours.
- functional_indicator_0h
- Source per-time-point functional indicator: 1 activating, -1 dampening/repressive, 0 not significant at FDR 0.05.
- functional_indicator_3h
- Source per-time-point functional indicator: 1 activating, -1 dampening/repressive, 0 not significant at FDR 0.05.
- functional_indicator_6h
- Source per-time-point functional indicator: 1 activating, -1 dampening/repressive, 0 not significant at FDR 0.05.
- functional_indicator_12h
- Source per-time-point functional indicator: 1 activating, -1 dampening/repressive, 0 not significant at FDR 0.05.
- functional_indicator_24h
- Source per-time-point functional indicator: 1 activating, -1 dampening/repressive, 0 not significant at FDR 0.05.
- functional_indicator_48h
- Source per-time-point functional indicator: 1 activating, -1 dampening/repressive, 0 not significant at FDR 0.05.
- functional_indicator_72h
- Source per-time-point functional indicator: 1 activating, -1 dampening/repressive, 0 not significant at FDR 0.05.
- functional_timepoint_count
- Number of seven time points with a nonzero source functional indicator.
Quality control
The authors required at least 80% of reads for a barcode to map to one sequence and at least three supporting reads, retained barcodes represented in both DNA and RNA, and reported 9,948 of 10,041 designed sequences with usable representation. Replicate activity was highly reproducible (mean Pearson correlation 0.98 per time point). For each perturbation method, the paper required four MPRAnalyze filters: significant PERT-versus-WT activity at one or more time points, significant PERT-versus-WT temporal behavior, WT or PERT activity above SCRAM controls, and WT or PERT temporal behavior different from SCRAM (FDR < 0.05). The consensus FRS set additionally required all four filters in method 3 and either method 1 or 2 with a consistent direction, yielding 598 rows. Package QC retained all 598/598 source FRS rows after requiring valid motif/region identifiers, a recognized effect code, valid motif coordinates/strand, and finite WT/PERT activity and log2 effect values at all seven time points.
Curation notes
This is one integrated lentiMPRA time-course experiment rather than seven independent libraries. The assay begins with H1/WA-01 human embryonic stem cells (Cell Ontology embryonic stem cell, CL:0002322) and transitions toward neural progenitors during the sampled window; the single biosample field therefore records the starting cultured cell type and the time course is described here. The processed table intentionally focuses on the uniform single-motif consensus FRS atlas (598 rows); raw GEO matrices and supplementary Dataset 3 retain the broader library and double-perturbation interaction data. Coordinates and the FRS source sheet are hg19, and source gene lists were normalized only from comma-delimited to semicolon-delimited cells.