Experiment / E2WRC60LHEpisomal Plasmid MPRA

CRE-seq activity of Crx-bound, unbound, mutant, and scrambled sequences in mouse retina

Massively parallel in vivo enhancer assay reveals that highly local features determine the cis-regulatory function of ChIP-seq peaks

A pooled library of 4,333 distinct 84-bp genomic or control CRE sequences, each represented by three independent 9-bp barcodes, was cloned upstream of a photoreceptor-specific Rhodopsin minimal promoter-DsRed reporter and electroporated into six explanted newborn mouse retina replicates. Reporter RNA barcode abundance was normalized to the plasmid DNA library to quantify sequence-level cis-regulatory activity.

Download processed table (CSV)Raw source data API documentation

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.

Organism: Mouse
Taxonomy ID: NCBITaxon:10090
Biosample: UBERON:0000966
Reference genome: NCBI37/mm9
Design focus: Region-focused
Region of interest: Not reported / not applicable

Perturbation & assay details

Basal / Untreated

CRE-seq reporter assay in explanted newborn mouse retina. The 84-bp test sequence was placed upstream of the Rhodopsin minimal promoter-DsRed reporter, with a 9-bp barcode in the reporter 3' UTR; six RNA replicates were measured and normalized to the starting plasmid DNA pool. The library included Crx ChIP-seq-bound regions with and without Crx motifs, matched unbound regions, A4C Crx motif mutants, and dinucleotide-preserving scrambled controls.

Processed data

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Visible columns (32 of 32)

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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 · 32 definitions

locus: Library locus identifier from the S1 sequence labels; Dataset S2 chromosome/start keys were normalized by adding one to the start coordinate for the join.
dataset_s2_locus: Exact LOCUS value from Dataset S2, retained for source traceability.
class: CRE class: CBRM (Crx-bound with motifs), CBRNO (Crx-bound without motifs), UBR (unbound with motifs), CBRMUT (Crx motif mutant), or scrambled controls SCRCBRM, SCRCBRNO, and SCRUBR.
sequence_84bp: The 84-bp CRE sequence extracted from positions 22-105 of the 150-mer oligo in Dataset S1; identical across barcodes for a locus.
activity_mean_rna_dna_ratio: Mean CRE-seq activity, calculated as RNA/DNA ratio and averaged across passing barcodes and six RNA replicates.
activity_log2_rna_dna_ratio: Log2 transformation of activity_mean_rna_dna_ratio; derived for convenience and blank if the ratio is nonpositive.
activity_sem: Standard error of the normalized CRE-seq activity mean.
activity_ci95: 95% confidence interval for the normalized CRE-seq activity mean.
activity_df: Degrees of freedom for the sequence-level activity estimate from author error propagation.
num_barcodes: Number of barcodes for the CRE passing the author plasmid-DNA filter (>1 read per million).
chip_reads: Number of Crx ChIP-seq reads assigned to the CBR; blank for non-CBR sequences.
crx_motif_count: Number of Crx motif occurrences above the P < 0.001 threshold.
crx_motif_score_sum: Sum of position-weight-matrix match scores for qualifying Crx motifs.
gc_fraction: Fraction of G and C nucleotides in the 84-bp CRE sequence.
orchid_sum: Sum of per-base ORChID2 minor-groove shape scores across the CRE.
photoreceptor_gene: Photoreceptor-gene association code from Corbo et al.; blank when not applicable.
crx_occupancy_mu6: Predicted Crx occupancy at medium Crx concentration (mu = 6).
crx_occupancy_mu9: Predicted Crx occupancy at high Crx concentration (mu = 9).
aaaa_count: Number of AAAA occurrences in the CRE sequence.
gc_dinucleotide_freq: Frequency of CC, CG, GG, and GC dinucleotides (source Dataset S2 field CCCGGGGC_FREQ).
nrl_motif_count: Number of Nrl motif occurrences above the P < 0.001 threshold.
nrl_motif_score: Sum of position-weight-matrix match scores for qualifying Nrl motifs.
mutation_pair_available: Whether Dataset S3 contains a matched CBRM/CBRMUT comparison for this locus; yes for the paired CBRM and CBRMUT rows.
mutant_fold_change: Dataset S3 FOLD_CHANGE, defined as mean CBRMUT activity divided by mean CBRM activity for the matched locus; repeated on both rows of the pair.
mutant_log2_fold_change: Log2 transformation of mutant_fold_change.
mutant_fold_se: Standard error associated with the WT/mutant fold-change comparison from Dataset S3.
mutant_welch_t: Welch's t statistic for the matched WT/mutant activity comparison.
mutant_df: Degrees of freedom for the matched WT/mutant Welch test.
mutant_n: Total replicate measurements used for each WT/mutant pair comparison.
mutant_raw_pval: Unadjusted P value for the matched WT/mutant comparison.
mutant_adj_pval: Benjamini-Hochberg adjusted P value for the matched WT/mutant comparison.
mutation_photoreceptor_gene: Photoreceptor-gene association code for the matched WT/mutant pair from Dataset S3.

Quality control

The authors discarded barcode reads that did not exactly match designed barcode sequences and retained only barcodes with plasmid DNA abundance greater than 1 read per million. Dataset S2 already summarizes only CREs with at least one passing barcode; the processed table retains rows with NUM_BC >= 1 and finite MEAN, SEM, and CI_95 values, with no additional activity threshold. The paper reports a mean Pearson correlation of 0.95 for barcode activity across biological replicates.

Curation notes

One experiment is represented: six biological electroporation replicates of the same pooled plasmid CRE-seq library. Dataset S1 contains all 12,999 barcode-level oligos; Dataset S2 contains 4,304 sequence-level rows after the authors' barcode DNA filter, and those are the rows retained in table.csv. Dataset S2 LOCUS values are consistently one coordinate lower than the matching S1 sequence labels and S3 pair LOCUS values; the table preserves the exact Dataset S2 key in dataset_s2_locus and uses the normalized S1 label in locus. WT/mutant statistics from S3 are attached to the 777 matched loci (1,554 CBRM/CBRMUT rows). The study tests Crx motif mutants but is primarily a region/control enhancer library rather than a natural-variant study.

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