Experiment / E4XQVNOJJEpisomal Plasmid MPRA

Synthetic TF-binding-site grammar library (SYN) in RW4 mouse embryonic stem cells

Synthetic and genomic regulatory elements reveal aspects of cis-regulatory grammar in mouse embryonic stem cells

A plasmid MPRA tested 624 synthetic 2-mer, 3-mer, and 4-mer elements containing consensus SOX2, OCT4/POU5F1, KLF4, and ESRRB binding sites in all allowed orders and orientations. Each primary element was represented by eight unique barcodes and compared with basal-promoter controls.

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Organism: Mouse
Taxonomy ID: NCBITaxon:10090
Biosample: CVCL:6442
Reference genome: Not reported / not applicable
Design focus: Synthetic / Motif-focused
Region of interest: Not reported / not applicable

Perturbation & assay details

Basal / Untreated

pCF10 plasmid libraries carried 40–80 bp synthetic elements immediately upstream of a minimal Pou5f1 promoter and dsRed/SV40 UTR reporter. Three biological RNA replicates and a shared plasmid-DNA pool were sequenced on Illumina NextSeq; element activity is the mean retained-barcode RNA-RPM/DNA-RPM ratio normalized to basal-promoter controls.

Processed data

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Column dictionary · 28 definitions

element_id: Unique synthetic element or control identifier from Supplementary file 1B.
element_type: Primary synthetic element, basal-promoter control, or CMF-Scalar calibration control.
sequence: 5-prime-to-3-prime sequence of the tested synthetic insert from Supplementary file 1B.
sequence_length_bp: Length of the tested insert sequence in base pairs.
tfbs_order: Order/orientation token string used in the paper element identifier; f and r suffixes denote forward and reverse site orientations.
tfbs_orientation: Comma-separated forward/reverse orientation for each site in tfbs_order.
tfbs_count: Number of consensus pluripotency TF-binding sites in the synthetic element.
tfbs_factors: Comma-separated factor names corresponding to tfbs_order (OCT4/POU5F1, SOX2, KLF4, ESRRB).
element_class: 2mer, 3mer, or 4mer class from the paper summary; blank for controls.
barcode_count_design: Number of designed barcodes for the element in the library design.
barcode_count_qc: Number of barcodes retained after the paper thresholds (DNA >=10 and every RNA replicate >=3 raw reads).
dna_count_qc: Sum of raw plasmid-DNA barcode counts over retained barcodes.
rna_count_qc_rep1: Sum of raw RNA barcode counts over retained barcodes in RNA replicate 1.
rna_count_qc_rep2: Sum of raw RNA barcode counts over retained barcodes in RNA replicate 2.
rna_count_qc_rep3: Sum of raw RNA barcode counts over retained barcodes in RNA replicate 3.
activity_rep1: Element-level RNA-RPM/DNA-RPM activity normalized to the retained basal-promoter controls in RNA replicate 1.
activity_rep2: Element-level RNA-RPM/DNA-RPM activity normalized to the retained basal-promoter controls in RNA replicate 2.
activity_rep3: Element-level RNA-RPM/DNA-RPM activity normalized to the retained basal-promoter controls in RNA replicate 3.
activity_mean: Mean of the three raw-count-derived normalized replicate activities.
activity_sem_replicates: Standard error of the three replicate activity values.
log2_activity_mean: Base-2 logarithm of activity_mean.
published_activity_mean: Element_normalized_mean reported in Supplementary file 1C; blank for controls.
published_activity_sem_all: Element_norm_SEM_all reported in Supplementary file 1C; blank for controls.
wilcoxon_p_rep1: Paper Wilcoxon p-value for the element versus basal expression in RNA replicate 1.
wilcoxon_p_rep2: Paper Wilcoxon p-value for the element versus basal expression in RNA replicate 2.
wilcoxon_p_rep3: Paper Wilcoxon p-value for the element versus basal expression in RNA replicate 3.
bonferroni_0_05_pass: Paper Boolean flag for Bonferroni-adjusted p < 0.05.
bonferroni_0_01_pass: Paper Boolean flag for Bonferroni-adjusted p < 0.01.

Quality control

Authors required each barcode to have at least 3 raw reads in every RNA replicate and at least 10 raw reads in the plasmid-DNA pool before RPM-ratio calculation; they reported R² = 0.98–0.99 across SYN biological replicates and recovery of 624/624 primary SYN elements. Package QC retained 624/624 primary elements (each had at least one barcode passing these thresholds), plus the basal and CMF-Scalar control rows when they had retained barcodes.

Curation notes

The table recomputes replicate activities from the GEO barcode-count files using the paper’s RPM, DNA-normalization, basal-control normalization, and barcode thresholds; published element means/SEM and p-values are retained alongside those values. The supplement’s 12 CMF-Scalar controls are included as explicit control rows, while the primary 624 SYN rows are the elements used in the paper’s main summary.

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