Experiment / E4BUO215GEpisomal Plasmid MPRA

Minimal-polylinker autonomous-activity MPRA in mouse retinal explants

Information content differentiates enhancers from silencers in mouse photoreceptors

The same two 2,422-element libraries were recloned with a minimal 23-bp polylinker in place of the basal Rho promoter and assayed in explanted P0 CD-1 mouse retinas. Three biological replicates per library were quantified by reporter cDNA versus input-plasmid DNA barcode sequencing; activity above the DNA-normalized baseline was treated as autonomous activity.

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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 Rho promoter was replaced by a minimal 23-bp polylinker between each 164-bp library sequence and DsRed. RNA cDNA and input plasmid DNA barcodes were RPM-normalized, divided to obtain per-barcode expression, averaged over barcodes and then over three biological replicates. There was no basal-promoter construct in this condition. The authors defined autonomous activity as log2(RNA/DNA) > 0; the processed table applies the same criterion to the author mean expression after the 1e-2 pseudocount used for the Polylinker log2 values.

Processed data

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Visible columns (37 of 37)

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Column dictionary · 37 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 mean Polylinker reporter activity: barcode cDNA/DNA expression without basal Rho normalization.
activity_sem: Author standard error of the mean for Polylinker activity across the three biological replicates.
replicate_1_activity: Polylinker RNA/DNA activity for biological replicate 1.
replicate_2_activity: Polylinker RNA/DNA activity for biological replicate 2.
replicate_3_activity: Polylinker RNA/DNA activity for biological replicate 3.
activity_log2: Log2 Polylinker activity with the author's 1e-2 pseudocount.
autonomous_activity: Boolean using the author's criterion: log2(RNA/DNA) > 0.
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 (>86% of reads for Polylinker libraries). Barcodes with fewer than 50 raw counts in the DNA sample were removed; barcodes with fewer than 8 RPM in any cDNA sample were treated as below detection and set to zero in all samples. Barcode expression was then averaged over barcode and biological replicates. This package additionally retained only constructs with numeric author activity and three numeric biological-replicate values: 9,808 of 9,988 construct records passed (4,759 WT, 4,090 MUT-allCrxSites, 665 MUT-shape, and 294 scrambled controls). Missing activity rows were excluded; valid zero activity values were retained. The authors note that low scrambled-sequence expression and the lack of a basal construct prevented the same calibration assessment used for the Rho assay.

Curation notes

This condition tests autonomous reporter activity after removal of the Rho promoter; it is not a separate natural-variant library. The 150 scrambled controls are shared between the two libraries and have valid Polylinker measurements but lack 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 and supplementary occupancy/information-content files; the GEO barcode-count files are retained as the primary raw count inputs.

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