Synthetic 3′UTR GFP MPRA in primary CD8+ T cells
Learning the sequence code of protein expression in human immune cellsA 467-member synthetic 3′UTR library, with six repeats of each designed motif in a GFP reporter, was delivered by an integrating retroviral vector to primary CD8+ T cells from three healthy donors. The sorted GFP-high, GFP-low, and total GFP-positive fractions were quantified by genomic-DNA amplicon sequencing.
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
αCD3/αCD28 activation for 48h before retroviral transduction; IL-2/IL-15/IL-7 expansion
Primary CD8+ T cells from three healthy donors were activated with αCD3/αCD28 for 48h before retroviral transduction and expanded with IL-2, IL-15, and IL-7. The library contained 467 synthetic 200-nt 3′UTR oligos: six repeats of each designed motif, including two 7-nt validation motifs, with fixed cloning flanks. Transduction was maintained at 8–12% to minimize multiple integrations; the top and bottom 15% GFP-expressing cells and the full GFP-positive fraction were sorted. Read2 was trimmed by removing 39 5′ bases and 57 3′ bases, aligned to the UTR library with Bowtie2 local mode (--very-sensitive), and counted with htseq-count. The author-normalized source counts are scaled to 10,000 reads per library; high-versus-low log2 enrichment columns in this package are derived from those normalized values.
Retroviral, genomically integrating GFP–synthetic 3′UTR reporter library (pRETRO-SUPER_GFP; not lentiviral); cells were transduced, GFP-high/GFP-low populations were FACS-sorted, and integrated 3′UTR abundance was quantified from genomic-DNA amplicon sequencing.
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 · 49 definitions
- element_id
- Unique source oligo identifier for the tested synthetic 3′UTR element.
- motif_sequence
- Designed motif sequence encoded in the element identifier.
- motif_length_nt
- Length of the designed motif in nucleotides (6 or 7).
- library_design
- Synthetic library design class: six-repeat 6-mer motif or six-repeat 7-mer validation motif.
- reporter_oligo_sequence
- Complete 200-nt synthetic reporter oligo sequence from supplementary Data S4, including fixed flanking cloning sequence.
- designed_repeat_count
- Number of intended motif repeats in the synthetic oligo (six).
- motif_occurrence_count_in_full_oligo
- Literal non-overlapping occurrences of the motif in the full 200-nt oligo; this can exceed the designed repeat count when the fixed sequence contains the motif.
- a_count
- Number of adenines in the designed motif.
- c_count
- Number of cytosines in the designed motif.
- g_count
- Number of guanines in the designed motif.
- t_count
- Number of thymines in the designed motif.
- ag_count
- A+G count in the designed motif.
- ct_count
- C+T count in the designed motif.
- gc_count
- G+C count in the designed motif.
- ag_fraction
- AG count divided by motif length.
- ct_fraction
- CT count divided by motif length.
- gc_fraction
- GC count divided by motif length.
- raw_count_all_27_libraries
- Sum of raw read counts for this element across all 27 deposited samples; used for the 40-read source QC threshold.
- qc_pass
- Whether the element passed the package QC filters and was retained in the table.
- gfp_high_rep1_raw_count
- Raw genomic-DNA amplicon read count in the GFP-high fraction for replicate 1 (HEK batch 1 or T-cell donor 1).
- gfp_high_rep2_raw_count
- Raw genomic-DNA amplicon read count in the GFP-high fraction for replicate 2 (HEK batch 2 or T-cell donor 2).
- gfp_high_rep3_raw_count
- Raw genomic-DNA amplicon read count in the GFP-high fraction for replicate 3 (HEK batch 3 or T-cell donor 3).
- gfp_low_rep1_raw_count
- Raw genomic-DNA amplicon read count in the GFP-low fraction for replicate 1 (HEK batch 1 or T-cell donor 1).
- gfp_low_rep2_raw_count
- Raw genomic-DNA amplicon read count in the GFP-low fraction for replicate 2 (HEK batch 2 or T-cell donor 2).
- gfp_low_rep3_raw_count
- Raw genomic-DNA amplicon read count in the GFP-low fraction for replicate 3 (HEK batch 3 or T-cell donor 3).
- gfp_total_rep1_raw_count
- Raw genomic-DNA amplicon read count in the full GFP-positive fraction for replicate 1 (HEK batch 1 or T-cell donor 1).
- gfp_total_rep2_raw_count
- Raw genomic-DNA amplicon read count in the full GFP-positive fraction for replicate 2 (HEK batch 2 or T-cell donor 2).
- gfp_total_rep3_raw_count
- Raw genomic-DNA amplicon read count in the full GFP-positive fraction for replicate 3 (HEK batch 3 or T-cell donor 3).
- gfp_high_rep1_normalized_count_10k
- Author-normalized GFP-high read count for replicate 1, scaled to 10,000 library reads.
- gfp_high_rep2_normalized_count_10k
- Author-normalized GFP-high read count for replicate 2, scaled to 10,000 library reads.
- gfp_high_rep3_normalized_count_10k
- Author-normalized GFP-high read count for replicate 3, scaled to 10,000 library reads.
- gfp_low_rep1_normalized_count_10k
- Author-normalized GFP-low read count for replicate 1, scaled to 10,000 library reads.
- gfp_low_rep2_normalized_count_10k
- Author-normalized GFP-low read count for replicate 2, scaled to 10,000 library reads.
- gfp_low_rep3_normalized_count_10k
- Author-normalized GFP-low read count for replicate 3, scaled to 10,000 library reads.
- gfp_total_rep1_normalized_count_10k
- Author-normalized full GFP-positive read count for replicate 1, scaled to 10,000 library reads.
- gfp_total_rep2_normalized_count_10k
- Author-normalized full GFP-positive read count for replicate 2, scaled to 10,000 library reads.
- gfp_total_rep3_normalized_count_10k
- Author-normalized full GFP-positive read count for replicate 3, scaled to 10,000 library reads.
- gfp_high_mean_normalized_count_10k
- Arithmetic mean of the three author-normalized GFP-high counts.
- gfp_high_sd_normalized_count_10k
- Sample standard deviation of the three author-normalized GFP-high counts.
- gfp_low_mean_normalized_count_10k
- Arithmetic mean of the three author-normalized GFP-low counts.
- gfp_low_sd_normalized_count_10k
- Sample standard deviation of the three author-normalized GFP-low counts.
- gfp_total_mean_normalized_count_10k
- Arithmetic mean of the three author-normalized full GFP-positive counts.
- gfp_total_sd_normalized_count_10k
- Sample standard deviation of the three author-normalized full GFP-positive counts.
- log2_enrichment_high_vs_low_rep1
- Derived log2(GFP-high normalized count / GFP-low normalized count) for replicate 1.
- log2_enrichment_high_vs_low_rep2
- Derived log2(GFP-high normalized count / GFP-low normalized count) for replicate 2.
- log2_enrichment_high_vs_low_rep3
- Derived log2(GFP-high normalized count / GFP-low normalized count) for replicate 3.
- log2_enrichment_high_vs_low_mean
- Arithmetic mean of the three replicate-specific high-versus-low log2 enrichments.
- log2_enrichment_high_vs_low_sd
- Sample standard deviation of the three replicate-specific high-versus-low log2 enrichments.
- log2_enrichment_high_vs_low_se
- Standard error of the mean high-versus-low log2 enrichment, calculated as sample SD divided by sqrt(3).
Quality control
Author QC trimmed read2, aligned with Bowtie2 --very-sensitive, counted with htseq-count, excluded motifs with fewer than 40 reads across all samples, and normalized each library to 10,000 reads. Package QC additionally required a unique element ID, a matching 200-nt oligo sequence, complete high/low/total counts for all three replicates, finite positive normalized values, and at least 40 raw reads summed across all 27 deposited libraries. All 467/467 library elements passed (minimum all-library total 315); no rows were removed.
Curation notes
Three primary CD8+ T-cell donors are represented as replicate 1–3 (GEO high/low/total triplets: GSM8840945/GSM8840954/GSM8840955, GSM8840956/GSM8840957/GSM8840958, and GSM8840959/GSM8840960/GSM8840961). This package treats the shared library in this biosample as one child experiment so each experiment table has one cell context. The high and low columns are FACS fractions rather than RNA/DNA ratios: high and low are the top and bottom 15% of GFP-positive cells; total is the full GFP-positive fraction. Normalized counts are the author/GEO 10,000-read values and include a pseudocount (source raw zeros are positive in the normalized table). log2_enrichment_high_vs_low_* is derived per replicate from these normalized counts. The full oligo sequence is 200 nt and contains six designed motif repeats; some motifs occur an additional time in fixed flanking sequence, so both designed_repeat_count and motif_occurrence_count_in_full_oligo are retained. Source rows from supplementary Data S4 and GEO matched exactly for all 467 elements and 27 samples. The assay is classified as Other because the paper specifies a genomically integrating retroviral vector, not lentivirus.