Focused 3′-UTR 8-mer screen with GFP-bin sequencing
High-throughput discovery of post-transcriptional cis-regulatory elementsA focused library of 3,918 selected 8-nucleotide elements, including pilot candidates, known miRNA and RNA-binding-protein motifs, conserved elements, reverse complements, and controls, was integrated in the human IQGAP1 3′UTR reporter in Flp-In T-REx 293 cells. FACS separated five GFP-intensity bins under a dsRed transcriptional gate, and matched DNA and reporter-RNA sequencing quantified regulatory activity and RNA abundance.
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.
Perturbation & assay details
Basal / Untreated
The library was delivered by FLP/FRT recombinase-mediated site-specific integration into a defined single-copy locus of Flp-In T-REx 293 cells. GFP was driven by EF-1α and the variable 8-mer was placed in the human IQGAP1 3′UTR; a PGK-dsRed reporter controlled for transcriptional noise. GFP+ cells within the middle 50% of dsRed intensity were sorted into five GFP bins (0–10%, 20–30%, 40–60%, 70–80%, and 90–100%), with replicate sorts and a middle-25% dsRed background collection. The variable 8-mer region was sequenced from integrated genomic DNA and poly(dT)-primed reporter cDNA on an Illumina HiSeq 2500 using 50-nt reads. The published score weights the five GFP bins from −2 to +2; the matched DNA/RNA measurements provide a log2 RNA/DNA proxy for steady-state RNA abundance and mRNA stability.
Processed data
50 rows per page. Click a cell to inspect its full value.
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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 · 21 definitions
- element_id
- Stable package identifier for the focused-screen 8-mer.
- sequence
- The tested 8-nucleotide sequence in the reporter 3′UTR.
- library_context
- Fixed human IQGAP1 3′UTR sequence context used for the variable 8-mer.
- score
- Published semi-quantitative GFP activity score from −2 (maximally repressive) to +2 (maximally activating), calculated from weighted GFP-bin RPM values.
- gfp_0_10_rpm
- Normalized reads per million from the lowest-GFP (0–10%) sorted population.
- gfp_20_30_rpm
- Normalized reads per million from the 20–30% GFP sorted population.
- gfp_40_60_rpm
- Normalized reads per million from the middle-GFP (40–60%) sorted population.
- gfp_70_80_rpm
- Normalized reads per million from the 70–80% GFP sorted population.
- gfp_90_100_rpm
- Normalized reads per million from the highest-GFP (90–100%) sorted population.
- dna_middle_50_rpm
- Normalized DNA reads per million from the middle-50% dsRed background population used as the GFP-bin enrichment denominator.
- gfp_0_10_enrichment
- 0–10% GFP enrichment relative to the DNA middle-50% dsRed background.
- gfp_20_30_enrichment
- 20–30% GFP enrichment relative to the DNA middle-50% dsRed background.
- gfp_40_60_enrichment
- 40–60% GFP enrichment relative to the DNA middle-50% dsRed background.
- gfp_70_80_enrichment
- 70–80% GFP enrichment relative to the DNA middle-50% dsRed background.
- gfp_90_100_enrichment
- 90–100% GFP enrichment relative to the DNA middle-50% dsRed background.
- dna_middle_25_rpm
- Normalized integrated-reporter DNA reads per million from the stringent middle-25% dsRed background population.
- rna_middle_25_rpm
- Normalized reporter-cDNA reads per million from the stringent middle-25% dsRed population.
- log2_rna_dna
- Published log2 ratio of reporter-cDNA RPM to integrated-reporter DNA RPM; a steady-state RNA-abundance proxy.
- selection_category
- Why the 8-mer was included in the focused library, such as pilot screen, conserved, miRNA target, RBP motif, or reverse complement.
- score_phenotype
- Published activity call: activating for score >1, repressive for score <−1, otherwise no effect.
- mrna_phenotype
- Published RNA-abundance call based on log2 RNA/DNA thresholds: destabilizing, stabilizing, or none.
Quality control
The authors filtered reads for a barcode, Phred quality scores greater than 20 in the barcode and variable 8-mer, and the correct CATAC and ATA flanking sequences; passing counts were normalized to sequencing-library size. The focused screen gated on the middle 50% of dsRed expression, required robust inclusion in a narrower middle-25% dsRed background set, and reported sorting-replicate concordance of Pearson r > 0.97. Package QC retained 2,004 of 2,005 rows in the supplied main-screen workbook: Score, DNA25 RPM, RNA25 RPM, and log2 RNA/DNA had to be finite, with both DNA25 RPM and RNA25 RPM greater than zero. The one excluded row (GTTTTGGA) had zero RNA25 RPM and an undefined log2 RNA/DNA value.
Curation notes
This is a targeted, integrated Sort-Seq/Flow-Seq reporter screen, not an allele-contrast experiment. The main workbook contains the author-filtered set of 8-mers with a stringent dsRed background measurement; the processed table removes only the single row whose RNA middle-25% count is zero, because its RNA/DNA ratio is undefined. The study reported 372 repressive and 461 activating elements using score cutoffs of <−1 and >1. The narrative reports 457 destabilizing elements, whereas Additional file 9 contains 478 rows with log2 RNA/DNA <−0.5; after excluding the zero-RNA row, the processed table contains 478 destabilizing and 417 stabilizing rows using the workbook's row-level calls and the published ±0.5 cutoffs. The table retains all other QC-passing elements, including those called no effect. The variable 8-mers are synthetic sequences in a fixed human IQGAP1 3′UTR context, so no individual genomic coordinates are assigned. The paper's auxiliary miRNA-seq mapping used hg19, which is why that assembly is recorded here. The exact Flp-In-T-REx-293 cell line was resolved to Cellosaurus CVCL:U427.