Experiment / E78ZIIY9EStandard STARR-seq

Methylation-sensitive enhancer/silencer reporter assay in T98G glioblastoma cells

Methylation-directed regulatory networks determine enhancing and silencing of mutation disease driver genes and explain inter-patient expression variation

A representative AK100 (library #100) target-enriched library of captured human glioblastoma regulatory fragments was cloned into a modified episomal self-transcribing reporter and assayed in T98G cells under unmethylated and in-vitro CpG-methylated plasmid conditions. The processed table contains the authors' significant 500-bp, 50%-overlapping reporter windows with paired counts, TAS values, and methylation-response annotations.

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Organism: Human
Taxonomy ID: NCBITaxon:9606
Biosample: CVCL:0556
Reference genome: hg19
Design focus: Region-focused
Region of interest: Not reported / not applicable

Perturbation & assay details

Unmethylated plasmid library versus in-vitro CpG-remethylated plasmid library using M.SssI

The modified pGL3-promoter episomal vector carried captured genomic segments inserted between AgeI-HF and SalI-HF downstream of the minimal promoter. Complete demethylation was obtained through bacterial propagation; the matched remethylated library was treated with NEB CpG methyltransferase M.SssI and checked by HpaII protection. Plasmid DNA and reporter cDNA were sequenced, and TAS was calculated as log2((RNA_j/DNA_j)/(RNA_total/DNA_total)); TAS < 0 denotes silencer activity and TAS > 0 denotes enhancer activity. GEO describes the library strategy as STARR-seq.

Processed data

50 rows per page. Click a cell to inspect its full value.

Visible columns (22 of 22)

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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 · 22 definitions

element_id: Stable package identifier formed from the source Table S7 element ID.
source_id: Element ID supplied in Table S7.
chrom: Chromosome of the source regulatory window.
start: Source Table S7 window start coordinate; retained exactly as supplied.
end: Source Table S7 window end coordinate; retained exactly as supplied and inclusive with start for a 500-bp span.
window_size_bp: Inclusive window size computed as end - start + 1; all retained rows are 500 bp.
dna_count_unmethylated: Reporter plasmid DNA read count in the unmethylated/demethylated condition.
rna_count_unmethylated: Reporter RNA read count in the unmethylated/demethylated condition.
tas_unmethylated: Source transcriptional activity score before methylation, calculated as log2((element RNA/DNA)/(total RNA/total DNA)).
p_value_unmethylated: Source chi-square p-value for the unmethylated-condition TAS against the total RNA/DNA ratio.
fdr_q_unmethylated: Source multiple-testing-adjusted q-value for the unmethylated-condition reporter activity test.
dna_count_methylated: Reporter plasmid DNA read count after in-vitro CpG methylation.
rna_count_methylated: Reporter RNA read count after in-vitro CpG methylation.
tas_methylated: Source transcriptional activity score after CpG methylation.
delta_tas_methylated_minus_unmethylated: Source TAS difference, equal to methylated TAS minus unmethylated TAS.
methylation_activity_fold_change: Derived methylated/unmethylated activity ratio, equal to 2 raised to delta TAS.
activity_class_unmethylated: Enhancer when unmethylated TAS is positive; silencer when it is negative.
activity_class_methylated: Enhancer when methylated TAS is positive; silencer when it is negative.
pre_methylation_qc_pass: Boolean indicating the published functional-element filter; all retained rows pass FDR q < 0.05 and unmethylated RNA count >= 100.
post_methylation_rna_qc_pass: Boolean indicating whether the methylated-condition RNA count is at least 100.
methylation_sensitive: Derived boolean; true when post-methylation RNA QC passes and the absolute TAS change is at least 1.5-fold on the activity scale (absolute delta TAS >= log2(1.5)).
methylation_response: Derived response class describing strengthening, weakening, or switching between enhancer and silencer activity; low post-methylation RNA and sub-threshold changes remain unclassified.

Quality control

Reads were aligned to hg19 with Bowtie2 using the first 40 bp from both ends; reads with mapping quality >40 aligned to probe targets were retained. Only on-target segments with at least one RNA read were included (623,223 pre-methylation and 304,998 post-methylation segment observations); >99% of targeted regions were present after bacterial propagation and re-extraction, and technical and biological sequencing replicates were performed. The paper defines functional elements as FDR q < 0.05 with at least 100 unmethylated-condition RNA reads. The packaged table retains 26,152 of 42,182 source windows, reproducing the reported 16,948 enhancers and 9,204 silencers; all retained rows have positive DNA and RNA counts in both methylation states.

Curation notes

The queried March 2021 bioRxiv preprint uses the earlier title Methylation-mediated retuning on the enhancer-to-silencer activity scale of networked regulatory elements guides driver-gene misregulation (DOI 10.1101/2021.03.02.433521); the package uses the open-access 2023 peer-reviewed version and its updated title. Supplementary Table S7 contains 42,182 unique 500-bp windows. Applying the paper's stated FDR q < 0.05 and unmethylated RNA >= 100 filter yields 26,152 rows, matching the reported 16,948 enhancers and 9,204 silencers; 728 retained rows have methylated RNA < 100 and are explicitly not called methylation-sensitive. The paper also has U87 GEO count files, but the scored reporter result presented in Table S7 and the Results is the T98G assay; U87 is represented separately from its public matrices in E6K8TIKZR. The study is region-focused and does not provide an allele-contrast/rsID MPRA library.

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