HEK293T 25-bp MAE-Seq fluorescence-selected enhancer library

A random 25-bp human genomic fragment library was cloned into a mini-promoter mCherry reporter plasmid and transfected into HEK293T cells. Fluorescence-selected cells were amplified and sequenced to identify active core enhancer loci; the released table is the 9,771-site positive enhancer catalog.

Processed tables are specific to each experiment. Column names, units, measurements, and table structure are not standardized across the database. Check this experiment’s column definitions and quality-control notes before comparing or combining data.

Organism: Human
Taxonomy ID: NCBITaxon:9606
Biosample: CVCL:0063
Reference genome: hg19
Design focus: Region-focused
Region of interest: Not reported / not applicable

Perturbation & assay details

Basal / Untreated

MAE-Seq used a plasmid reporter based on pMX-GFP, modified to carry a mini-promoter and mCherry (pMX-mP-mCherry). Synthetic random DNA fragments of 20–60 bp were evaluated, with 25 bp selected for the final library. HEK293T cells at 70–80% confluence were transfected with 4 micrograms library DNA per 1 million cells using FuGENE HD; fluorescent cells were collected 24 hours later, vector-flanking PCR amplified, and sequenced on an Illumina HiSeq 2000. This is a fluorescence-selection/output-library assay rather than a barcode-level RNA/DNA ratio assay.

Quality control

The authors trimmed adapters and homologous arms with cutadapt v1.16, retained reads in which more than 50% of bases had Phred quality >=20 using fastx-toolkit, removed duplicates with FastUniq, selected 25-bp reads, aligned them to hg19 with Bowtie2 v2.1.0 (-D 20 -R 3 -N 0 -L 17 -i S,1,0.50 --end-to-end), and retained uniquely mapped reads without insertions or deletions. The paper reports 7,412,991 raw reads, 7,412,979 after trimming, 7,406,623 after quality filtering, 532,017 after duplicate removal, 22,529 unique mapped reads, 12,412 deduplicated unique mapped reads, and 9,771 unique sites. Package QC removed the trailing empty line from the GEO annotation export; all 9,771 remaining records have valid 25-bp intervals and unique coordinates. Valid hg19 alternate/unplaced contigs were retained.

Curation notes

The sole GEO sample is GSM3713164 (N25 fragments), representing the fluorescence-selected output library. The public supplement is an annotated positive-site catalog, not a quantitative MPRA count matrix: no barcode-level DNA/RNA counts, allelic contrasts, log2 activity scores, or per-replicate measurements are available. The source Peak Score is 1 for every row and should be treated only as an annotation field. One valid 25-bp interval lacks a source PeakID and is retained under a package-generated element_id. Ten records map to valid hg19 alternate or unplaced contigs and are retained rather than discarded. HEK293T was resolved to Cellosaurus CVCL:0063.

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HEK293T 25-bp MAE-Seq fluorescence-selected enhancer library

Processed data

Quality control

Curation notes

Cite OpenMPRA

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