Experiment / E0DHJKB2UEpisomal Plasmid MPRA

Rho promoter CRX-targeted MPRA in mouse retinal explants

Information content differentiates enhancers from silencers in mouse photoreceptors

Two 2,422-element libraries of 164-bp mouse genomic sequences, CRX-site mutants, shape mutants, and scrambled controls were cloned upstream of the basal rod photoreceptor Rhodopsin (Rho) promoter and DsRed. Each library was electroporated into explanted P0 CD-1 mouse retinas in three biological replicates, harvested at P8, and quantified by reporter cDNA versus input-plasmid DNA barcode sequencing.

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Organism: Mouse
Taxonomy ID: NCBITaxon:10090
Biosample: UBERON:0000966
Reference genome: mm10
Design focus: Region-focused
Region of interest: Not reported / not applicable

Perturbation & assay details

Basal / Untreated

The episomal constructs contain 164-bp genomic candidates upstream of the basal Rho promoter and a DsRed reporter. Each unique sequence was represented by three unique 9-bp barcodes; the basal-promoter-only control had 18 barcodes. RNA cDNA and input plasmid DNA barcodes were sequenced. Five retinas were pooled per biological replicate. Rho barcode expression was RPM-normalized, calculated as cDNA/DNA, normalized to replicate-specific basal Rho expression, and averaged across the three replicates. Enhancer, silencer, inactive, and ambiguous classes were assigned by the authors using Welch's t-tests, Benjamini-Hochberg FDR, and twofold thresholds relative to basal Rho activity.

Processed data

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Column dictionary · 43 definitions

element_id: Exact author construct label: mm10 interval, four-character library annotation code, and variant suffix; matches the FASTA and GEO count labels.
parent_element_id: Construct label with the variant suffix removed.
library: Author library number, 1 or 2.
variant: Construct sequence state: WT, MUT-allCrxSites, MUT-shape, or scrambled.
construct_role: Package label distinguishing ordinary library sequences from scrambled controls.
chrom: Chromosome from the mm10 interval encoded in the construct label.
coordinate_start: Start coordinate from the author mm10/BED-style construct label.
coordinate_end: End coordinate from the author mm10/BED-style construct label.
annotation_code: Four-character author code: CRX binding status, CRX motif status, photoreceptor ATAC-seq status, and histone-mark status.
crx_binding_annotation: Decoded first annotation character: CRX ChIP-seq peak or unbound in CRX ChIP-seq.
crx_motif_annotation: Decoded second annotation character: CRX PWM hit, Shape motif, or both.
atac_annotation: Decoded third annotation character: ATAC-seq peak in rods, cones, both, or neither of the specified photoreceptor samples.
histone_annotation: Decoded fourth annotation character: enhancer-marked, promoter-marked, H3K27ac-negative/H3K4me3-positive, or neither.
sequence_length_bp: Length of the tested DNA sequence in base pairs; all retained sequences are 164 bp.
sequence: Tested DNA sequence, 5-prime to 3-prime as supplied in the library FASTA.
activity_score_rna_dna: Author per-sequence Rho reporter activity: barcode cDNA/DNA expression after replicate-specific basal Rho normalization and averaging.
activity_sd: Author standard deviation of the Rho activity across the three biological replicates.
replicate_count: Number of biological replicates contributing to the author activity summary.
replicate_1_activity: Rho RNA/DNA activity for biological replicate 1 after the author's replicate-specific normalization.
replicate_2_activity: Rho RNA/DNA activity for biological replicate 2 after the author's replicate-specific normalization.
replicate_3_activity: Rho RNA/DNA activity for biological replicate 3 after the author's replicate-specific normalization.
activity_log2: Author log2 Rho activity, calculated with a 1e-3 pseudocount.
activity_pvalue: Author two-sided Welch's t-test p-value for difference from the basal Rho promoter.
activity_qvalue: Author Benjamini-Hochberg FDR-adjusted activity p-value.
activity_class: Author Rho activity class (Strong enhancer, Weak enhancer, Inactive, Silencer, or blank for ambiguous/unclassified); Scrambled control is a package label for controls.
wt_vs_mut_log2: Author log2 fold change of WT activity versus the all-CRX-site mutant for the parent element; repeated on the relevant construct rows and blank when unavailable.
wt_vs_mut_pvalue: Author Welch's t-test p-value for the WT versus all-CRX-site mutant activity comparison.
wt_vs_mut_qvalue: Author FDR-adjusted p-value for the WT versus all-CRX-site mutant activity comparison.
crx_bound: Author boolean indicating overlap with a CRX ChIP-seq peak.
nrl_bound: Author boolean indicating overlap with an NRL ChIP-seq peak.
mef2d_bound: Author boolean indicating overlap with a MEF2D ChIP-seq peak.
binding_group: Author category for the combination of CRX, NRL, and MEF2D ChIP-seq binding.
crx_predicted_occupancy: Author predicted CRX motif occupancy.
gfi1_predicted_occupancy: Author predicted GFI1 motif occupancy.
maz_predicted_occupancy: Author predicted MAZ motif occupancy.
mef2d_predicted_occupancy: Author predicted MEF2D motif occupancy.
ndf1_predicted_occupancy: Author predicted NDF1/NeuroD-family motif occupancy.
nrl_predicted_occupancy: Author predicted NRL motif occupancy.
rorb_predicted_occupancy: Author predicted RORB motif occupancy.
rax_predicted_occupancy: Author predicted RAX motif occupancy.
total_predicted_occupancy: Author sum of predicted occupancy for the eight analyzed transcription factors.
motif_diversity: Author number of analyzed transcription factors with predicted occupancy above 0.5.
information_content_bits: Author motif information content/entropy in bits, computed from predicted occupancies.

Quality control

Author QC retained reads whose barcode sequence exactly matched the designed sequence (>93% of reads for Rho libraries). Barcodes with fewer than 10 raw counts in the DNA sample were removed; barcodes with fewer than 5 raw counts in any cDNA sample were treated as below detection and set to zero in all samples. Barcode counts were RPM-normalized and averaged per sequence across barcode replicates and biological replicates. This package additionally retained only constructs with numeric author activity and three numeric biological-replicate values: 9,932 of 9,988 construct records passed (4,821 WT, 4,139 MUT-allCrxSites, 673 MUT-shape, and 299 scrambled controls). Missing activity rows were excluded; valid zero activity values were retained. No activity-significance filter was applied to the table, so valid inactive and ambiguous measurements remain available.

Curation notes

This is a region-focused genomic candidate library rather than a natural-allele or GWAS library; MUT-allCrxSites and MUT-shape are designed sequence perturbations. Pairwise WT-versus-mutant fields are available primarily for all-CRX-site mutants. The 150 scrambled controls in each library have valid Rho measurements but do not have the supplementary TF-binding/occupancy annotations, so those fields are blank. The processed table joins the author repository's per-library summaries with the supplied FASTA, supplementary activity/annotation, occupancy, and information-content files; the GEO barcode-count files are retained as the primary raw count inputs.

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