GEO series

Lentiviral single-cell MPRA of synthetic enhancers reveals motif affinity-based encoding of cell state specificity [sc-lentiMPRA 2]

GSE331030 Mus musculus; Homo sapiens Expression profiling by high throughput sequencing 66 samples Submitted 2026/05/29 Platform GPL30173Platform GPL21626Platform GPL30172

Summary

Cell state-specific gene expression programs emerge from the interplay between cis-regulatory elements (CREs), such as enhancers, and transcription factors (TFs). Massively parallel reporter assays (MPRAs) have enabled large-scale dissection of CRE function, but bulk approaches cannot resolve cell state specificity on continuous trajectories of cellular differentiation, and existing single-cell MPRAs are not readily applicable to primary cell differentiation models. Here, we developed a single-cell lentiviral Massively Parallel Reporter Assay (sc-lentiMPRA) that overcomes these limitations and enables parallel quantification of enhancer activity and cellular transcriptome. Applying sc-lentiMPRA in blood stem differentiation, we profiled the activity and specificity of ~160 fully synthetic enhancers with controlled motif composition and affinities across ~190,000 single cells. Focusing on Trp53 and Cebpa, we show that enhancers with high and low affinity motifs differ qualitatively and quantitatively in their responses to TF expression gradients. For Trp53, low-affinity motifs exhibited near-linear correlation with TF expression, whereas high-affinity motifs showed reduced sensitivity to TF levels and a potential contribution of cofactor availability. In contrast, Cebpa-associated enhancers displayed non-linear behaviors. Together, sc-lentiMPRA establishes a powerful framework for systematically relating enhancer architecture and TF expression to regulatory output at single-cell resolution during cellular differentiation.

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Direct links to NCBI, no account and no request form: the whole study as GSE331030_RAW.tar, processed values as the series matrix, the supplementary file directory, and per-sample supplementary files for any of the 66 samples. Raw sequencing reads are also available from ENA.

Whole study · RAW.tar ↓ Series matrix ↓ All supplementary files ↗ Raw reads on ENA ↗

Also filed as BioProject PRJNA1466561 and SRA study SRP700696. Searching any of these in the dataset finder brings you back here.

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Study design

5 conditions, mostly with about 8 replicates each

mHSC sc-lentiMPRA Library Beta, GFP quantitat… ×12 mHSC sc-lentiMPRA Library Alpha, GFP quantita… ×8 mHSC sc-lentiMPRA Library Alpha, guide presen… ×8 mHSC sc-lentiMPRA Library Alpha, TAP gene exp… ×8 mHSC sc-lentiMPRA Library Beta, guide presenc… ×4

Supports a between-group comparison across 40 samples.

5 replicated groups read from the first 40 of 66 sample titles; they account for 40 of them. Check it against the sample list below before relying on it.

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