An episomal synSTARR-seq library varied five consecutive flanking nucleotides around a fixed Cgt glucocorticoid receptor binding sequence, representing 1,024 designed flank variants. U2OS-GR cells were treated with 1 µM dexamethasone or 0.1% ethanol vehicle for 4 h in three biological replicates per condition, and all 1,024 released variant-level results passed package QC.
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Organism
Human
Taxonomy ID
NCBITaxon:9606
Biosample
CVCL:0042
Reference genome
Not reported / not applicable
Design focus
Synthetic / Motif-focused
Region of interest
Not reported / not applicable
Perturbation & assay details
1 µM dexamethasone for 4 h versus 0.1% ethanol vehicle control
Synthetic STARR-seq (synSTARR-seq) used synthetic oligos cloned into an episomal human STARR-seq reporter downstream of a minimal promoter, so active inserts were quantified through self-transcribing reporter RNA. The Cgt library contains a fixed 15 bp context followed by five randomized flank bases. Five million U2OS-GR cells were nucleofected with 5 µg library DNA, treated the next day with 1 µM dexamethasone or 0.1% ethanol for 4 h, and sequenced using Illumina 50 bp paired-end reads. Reads were retained only when they exactly matched the expected synthetic insert length and nucleotide composition; duplicate reads were intentionally retained for quantitative sequence-level counting. DESeq2 compared dexamethasone and vehicle counts across three biological replicates per condition.
Processed data
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Column dictionary · 12 definitions
element_id
Unique package identifier for the synthetic Cgt flank sequence.
Package-derived call: significant_activation when adjusted_p_value < 0.01 and log2_fold_change > 0, significant_negative_effect when adjusted_p_value < 0.01 and log2_fold_change < 0, otherwise not_significant.
Quality control
The authors filtered reads to exact insert matches, retained duplicate reads, and identified differential sequence activity with DESeq2 using adjusted p-value < 0.01. Package QC required a 20 bp A/C/G/T-only sequence with a 5 bp variable segment, unique sequence identifiers, finite baseMean/log2FoldChange/lfcSE/stat/pvalue/padj values, positive baseMean, p-value and adjusted p-value in [0,1], and nonnegative lfcSE. All 1,024 of 1,024 released rows passed these checks and were retained; no additional abundance cutoff was imposed on the flank library.
Curation notes
The ArrayExpress source file All_Cgt-flanks_DESeq2.tab omits the leading sequence name from its header; conversion treated the first token of each data row as sequence and preserved the remaining seven source fields. CVCL:0042 is the parental human U2OS osteosarcoma line; the assayed U2OS-GR derivative stably expresses rat GRα and has no separate Cellosaurus accession identified. The paper’s Cgt flank results classify 189 variants as significantly enhancing and 125 as significantly blunting at adjusted p-value < 0.01, with the remainder neutral by that criterion.