Experiment / E44ZNZ067Integrated lentiMPRA

HSPC primary-cell lentiMPRA — Library C broad pairwise motif screen

Design principles of cell-state-specific enhancers in hematopoiesis

Library C sampled a broad space of pairwise combinations covering 42 transcription factors, including different motif affinities, orientations, spacings, and arrangements in random DNA. The library was measured in seven differentiated murine HSPC progenitor states.

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Organism: Mouse
Taxonomy ID: NCBITaxon:10090
Biosample: CL:0000037
Reference genome: Not reported / not applicable
Design focus: Synthetic / Motif-focused
Region of interest: Not reported / not applicable

Perturbation & assay details

Basal / Untreated (ex vivo HSPC differentiation)

Lentiviral reporter assay using pLS-SceI (Addgene plasmid #137725), with the synthetic or genomic regulatory element cloned upstream of a minimal promoter and an EGFP reporter. Primary murine Lin−Kit+ HSPCs were infected, cultured for ex vivo differentiation, FACS-sorted into progenitor states, and assayed by DNA/RNA UMI counts from the reporter 5′ UTR. The two replicate activities are log2 library-size-normalized RNA/DNA values; random-DNA subtraction and Trp53 scaling follow the authors' published definitions.

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 · 31 definitions

element_id: Unique identifier for the tested gene-regulatory element (source CRS).
source_data_object: Name of the main DATA object in the public Figshare R archive.
cell_state_id: Source cell-state cluster identifier; aggregate_HSPC denotes the supplied across-cell-state aggregate view.
cell_state: Cell-state label mapped from the archived cellstate.map vector; K562 denotes State_9K.
library: Paper/library identity assigned from the public DATA-object name and the article (A–H).
source_library_label: Verbatim Library field in the R archive, retained for provenance; it is inconsistent with the object/paper identity for some libraries.
sequence: Reporter insert DNA sequence; in synthetic libraries uppercase letters encode placed motifs and lowercase letters encode background DNA as supplied.
rna_count_rep1: Raw UMI-derived reporter RNA molecule count for replicate 1.
dna_count_rep1: Raw UMI-derived plasmid DNA molecule count for replicate 1.
rna_count_rep2: Raw UMI-derived reporter RNA molecule count for replicate 2.
dna_count_rep2: Raw UMI-derived plasmid DNA molecule count for replicate 2.
rna_normalized_rep1: Library-size-normalized reporter RNA count for replicate 1.
dna_normalized_rep1: Library-size-normalized plasmid DNA count for replicate 1.
rna_normalized_rep2: Library-size-normalized reporter RNA count for replicate 2.
dna_normalized_rep2: Library-size-normalized plasmid DNA count for replicate 2.
activity_log2_raw_rep1: Raw log2 RNA/DNA activity for replicate 1 after library-size normalization.
activity_log2_raw_rep2: Raw log2 RNA/DNA activity for replicate 2 after library-size normalization.
activity_log2_adjusted_rep1: Replicate-1 activity after subtracting the median random-DNA baseline.
activity_log2_adjusted_rep2: Replicate-2 activity after subtracting the median random-DNA baseline.
activity_log2_raw_mean: Mean raw log2 RNA/DNA activity across the two replicates.
activity_log2_adjusted_mean: Mean baseline-adjusted log2 RNA/DNA activity across the two replicates; preferred quantitative activity score.
activity_scaled_mean: Source visualization scale with random-DNA activity at 0 and the Trp53 reference at 1; not the preferred score for modeling or statistical testing.
motif_spacing_bp: Spacing between placed motif sites in base pairs (source spacer).
tf1_name: Name of the first TF motif from the 5′ end of the sequence.
tf1_affinity: Designed affinity of the first TF motif/site set.
tf1_orientation: Orientation of the first TF motif/site set.
tf2_name: Name of the second TF motif from the 5′ end of the sequence.
tf2_affinity: Designed affinity of the second TF motif/site set.
tf2_orientation: Orientation of the second TF motif/site set.
sites_per_factor: Number of sites assigned to each factor in the pair design (source TFnumber).
tf_site_order: Arrangement of paired sites, such as Alternate or Block (source TForder).

Quality control

The authors filtered the GRE–barcode association by alignment score (290–292), dominant assignment support (assigned reads at least five times deviant-assignment reads), barcode homopolymer content (removed barcodes with more than 10 identical nucleotides), and sequencing-error correction. MPRA reads required concordant forward/reverse barcode reads and underwent UMI error correction. The supplied DATA frame is the authors' post-processing/post-QC element-by-cell-state table. Package QC additionally required complete identifiers, non-empty sequences, positive raw DNA/RNA counts, and finite normalized/activity values. Public processing parameters for HSPC Library C were: minimum two reads for GRE–barcode association, UMI read threshold 1, cap DNA UMI counts, count RNA-only barcodes on DNA, minimum 100 UMIs per GRE, and DNA normalization. The public DATA object retained 163,683 of 163,683 rows (0 removed); the pooled adjusted-activity replicate Pearson correlation is 0.626.

Curation notes

The DATA object is HSPC.libC and its pair-design fields identify the paper's Library C. Its verbatim source_library_label is LibB, which is retained for provenance; the paper/object identity is used for the library field. The pooled correlation is lower than the article's per-state summary because it combines all states and constructs.

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