Experiment / E6L0BPURLEpisomal Plasmid MPRA

Round 3 motif-centric and random-control MPRA

Active learning of enhancer and silencer regulatory grammar in photoreceptors

Four episomal MPRA sublibraries tested motif-centric insertions, motif scrambles, swaps, high-confidence pilot sequences, and randomized active-learning controls derived from the Round 1 training space. The libraries were assayed in P0 CD-1 mouse retinal explants with the Rho basal promoter reporter.

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

Perturbation & assay details

Basal / Untreated

Oligo-synthesized 164-bp test sequences with unique 9-bp cis-regulatory barcodes were cloned upstream of the rod-specific Rhodopsin (Rho) basal promoter driving DsRed in episomal pJK03-derived reporter plasmids. P0 CD-1 mouse retinas were electroporated, five retinas were pooled per biological replicate, and tissue was cultured for eight days; barcode RNA/cDNA counts were normalized to input plasmid DNA and basal or scrambled pseudobasal controls as specified by the authors.

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 · 46 definitions

element_id: Unique row identifier copied from the authors’ processed activity table.
label: Original sequence/construct label, including genomic coordinates and perturbation suffixes when present.
library: MPRA library name assigned by the authors (library1 through library9b).
data_batch_name: Authors’ active-learning batch annotation, such as Genomic, Round2, Round3a, Round4a, or Round4b.
sequence: 164-bp DNA test sequence used in the reporter construct.
sequence_length: Length of sequence in nucleotides; package QC required 164.
gc_fraction: Fraction of sequence bases that are G or C.
activity_score: Author basal-normalized mean MPRA activity on a linear scale; expression = 1 corresponds to basal activity.
activity_score_std: Author basal-normalized standard deviation across RNA replicate activity values.
activity_replicates: Number of RNA replicate activity values contributing to the author summary.
activity_log2: Log2 of the author basal-normalized MPRA activity score.
p_value_vs_basal: Author Welch-test p-value comparing the element with basal activity.
q_value_vs_basal: Author FDR-adjusted q-value for the comparison with basal activity.
lognormal_mu: Author fitted log-normal location parameter for replicate activity values.
lognormal_sigma: Author fitted log-normal scale parameter for replicate activity values.
activity_class: Author-discretized activity class: Silencer, Inactive, WeakEnhancer, or StrongEnhancer.
dna_count_sum: Sum of raw input-plasmid barcode counts assigned to this element across all matching barcodes.
rna_count_sum_rep1: Sum of raw RNA barcode counts for the first RNA sample in the library.
rna_count_sum_rep2: Sum of raw RNA barcode counts for the second RNA sample in the library.
rna_count_sum_rep3: Sum of raw RNA barcode counts for the third RNA sample in the library.
rna_count_sum_rep4: Sum of raw RNA barcode counts for the fourth RNA sample in the library, when available.
rna_count_sum_rep5: Sum of raw RNA barcode counts for the fifth RNA sample in the library, when available.
rna_count_sum_mean: Arithmetic mean of available raw RNA count sums across replicates.
rna_count_sum_cv: Population coefficient of variation of available raw RNA count sums; this is a package-level context metric, not the barcode-level author QC CV.
rna_nonzero_reps: Number of RNA samples with at least one matching raw count.
immediate_precursor: Sequence label from which the current perturbation was directly derived, when annotated by the authors.
original_seq: Original genomic sequence identifier associated with a perturbation, when annotated.
standard_seq: Boolean flag for an assay standard sequence.
scrambled: Boolean flag for a scrambled negative-control sequence.
rational_mutagenesis: Boolean flag for a hand-designed/rational motif perturbation.
ic_scan: Boolean flag for an information-content scan sequence.
original_genomic: Boolean flag for an original genomic sequence in libraries 1–2.
mut_all_crx: Boolean flag for a sequence with all CRX motifs mutated.
mut_shape: Boolean flag for a sequence with a CRX shape motif mutation.
entropy_sampling: Boolean flag for selection by entropy uncertainty sampling.
margin_sampling: Boolean flag for selection by margin uncertainty sampling.
random_sampling: Boolean flag for a random active-learning control.
high_conf_pilot: Boolean flag for a high-confidence pilot prediction sequence.
high_conf_cnn: Boolean flag for a high-confidence CNN prediction sequence.
l9_controls: Boolean flag for known-control sequences included in library 9.
l9_repeat_l8: Boolean flag for library 8 sequences repeated in library 9.
test_set: Boolean flag for the motif-mutagenesis test set.
derived_from_test_set_seq: Boolean flag for a sequence derived from a held-out test-set sequence.
cnn_validation_set: Boolean flag for membership in the CNN validation set.
svm_train: Boolean flag indicating inclusion in the authors’ SVM training set.
cnn_train: Boolean flag indicating inclusion in the authors’ CNN training set.

Quality control

Author QC required exact cBC/mBC context matches, at least 50 input-plasmid DNA counts per barcode, and RNA barcode coefficient of variation no greater than 0.8 across cDNA samples; counts were depth-normalized, RNA was normalized to plasmid DNA, barcodes were averaged to CRE activity, and activity was basal-normalized (or scrambled-pseudobasal-normalized when basal recovery was poor). The author-published summary retained only sequence measurements with more than one RNA replicate. Package QC additionally required unique element IDs, 164-bp A/C/G/T-only sequences, finite author activity statistics, at least one matching raw DNA count, and at least two nonzero raw RNA replicate totals. Retained 32333 of 32333 source rows; removed 0 rows: none.

Curation notes

Libraries4–7 contain multiple author-assigned batches (Round3a, Round3b, Round3c, test/validation material, and controls); data_batch_name and the boolean annotations should be used to separate these subsets. Round3a and Round3b are co-represented in libraries4–5, while Round3c is represented in libraries6–7.

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