Experiment / E156A6C2EStandard STARR-seq

Baboon mSTARR-seq methylation-dependent enhancer activity screen in K-562 cells

DNA methylation signatures of early-life adversity are exposure-dependent in wild baboons

A baboon-specific mSTARR-seq library of 300–800 bp genomic fragments generated by mechanical shearing and MspI digestion was split into fully CpG-methylated and sham/unmethylated pools and transfected into human K-562 cells. Six biological replicates per methylation state were measured by targeted insert DNA-seq and plasmid-specific RNA-seq, and activity was modeled in 500-bp genomic windows.

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Organism: NCBITaxon:9555
Taxonomy ID: NCBITaxon:9555
Biosample: CVCL:0004
Reference genome: Panubis1.0 (GCA_008728515.1)
Design focus: Region-focused
Region of interest: Not reported / not applicable

Perturbation & assay details

Fully CpG-methylated insert library produced with M.SssI versus sham/unmethylated insert library treated with water; 48 h post-transfection

Methyl-STARR-seq (mSTARR-seq) using the CpG-free pmSTARRseq1 plasmid backbone. Genomic DNA from Papio anubis individual #15944, the source of the Panubis1.0 assembly, was fragmented by Covaris S220 shearing or MspI digestion, size-selected at 300–800 bp, cloned by Gibson assembly, and transformed into GT115 E. coli. Matched pools were treated with M.SssI or water, 40 µg of each pool was chemically transfected into approximately 20 million K-562 cells, and cells were harvested after 48 h for targeted paired-end DNA-seq of plasmid input and plasmid-specific RNA-seq of enhancer-like transcripts. MspI-derived and sheared fragments were counted separately over discrete 500-bp Panubis1.0 windows before the authors’ source-preference rule was applied for the packaged table.

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 · 25 definitions

chromosome: Panubis1.0 chromosome or contig for the tested window.
start: Reported start coordinate of the 500-bp window.
end: Reported end coordinate of the 500-bp window.
window_id: Author site identifier in the form chromosome_start_end.
window_length_bp: end minus start; the assay targets 500-bp windows, with three author-supplied source intervals shorter than 500 bp retained and flagged in the package comments.
p_intercept: Model p-value for the intercept in the authors’ limma-voom nested model.
p_methylation_treatment: Model p-value for the methylation-treatment main effect in the DNA-reference design.
p_rna_dna_unmethylated: P-value for the RNA-versus-DNA effect in the sham/unmethylated condition.
p_rna_dna_methylated: P-value for the RNA-versus-DNA effect in the fully methylated condition.
beta_methylation_treatment: Estimated methylated-versus-sham treatment effect in the DNA-reference design.
beta_rna_dna_unmethylated: Estimated RNA-versus-DNA activity effect for the sham/unmethylated condition; positive values indicate more RNA than expected from DNA input.
beta_rna_dna_methylated: Estimated RNA-versus-DNA activity effect for the fully methylated condition; positive values indicate more RNA than expected from DNA input.
se_beta_methylation_treatment: Standard error of beta_methylation_treatment.
se_beta_rna_dna_unmethylated: Standard error of beta_rna_dna_unmethylated.
se_beta_rna_dna_methylated: Standard error of beta_rna_dna_methylated.
beta_methylation_interaction: Methylation-dependent activity contrast, calculated as beta_rna_dna_methylated minus beta_rna_dna_unmethylated.
se_beta_methylation_interaction: Standard error reported for the methylation interaction contrast.
t_methylation_interaction: T statistic for the methylation interaction contrast.
p_methylation_interaction: P-value for the methylation interaction contrast.
fdr_rna_dna_methylated: Permutation-based FDR/q-value for the methylated RNA-versus-DNA activity test; NA indicates that the row was not in the authors’ positive-activity candidate subset.
fdr_rna_dna_unmethylated: Permutation-based FDR/q-value for the sham/unmethylated RNA-versus-DNA activity test; NA indicates that the row was not in the authors’ positive-activity candidate subset.
fdr_methylation_interaction: Permutation-based FDR/q-value for the methylation-dependent activity interaction; NA indicates that the interaction was not assessed for that row.
regulatory_activity_call: Author-supplied call: sham for significant activity in the unmethylated/sham state, meth for significant activity in the methylated state, none for positive-activity candidates not significant in either state, and NA when the positive-activity FDR was not applicable.
methylation_dependence_call: Author-supplied condition label for a significant methylation-dependent activity call (sham or meth); NA means no significant call was reported.
library_source: Fragment-library source retained for the row: MspI for the MspI-digested library or sheared for the mechanically fragmented library. MspI was preferred on source overlap per the paper.

Quality control

Author QC trimmed reads with Cutadapt/Trim Galore, mapped them to Panubis1.0 with bwa, retained properly paired reads with MAPQ ≥10, classified MspI-derived versus sheared fragments by MspI cut-site sequence, and counted overlaps with discrete 500-bp windows. A window was retained when median coverage was ≥4× in both methylated and sham DNA treatments, DNA counts were nonzero in at least 4 of 6 replicates in both treatments, and RNA counts were nonzero in at least 4 of 6 replicates in either treatment. This produced 210,942 MspI and 41,521 sheared source-specific analyzable rows. Library-size normalization used edgeR and RNA-versus-corresponding-DNA normalization used limma voomWithQualityWeights; regulatory and methylation-dependence calls used 100 paired-label permutations with a 10% FDR cutoff. Packaging retained all source-specific QC-passing windows while selecting the MspI row where a window occurred in both sources, as stated by the authors; the resulting table contains 241,256 unique windows (210,942 MspI and 30,314 sheared-only rows).

Curation notes

The raw pnas.2309469121.sd05.txt file contains 252,463 source-specific rows (210,942 MspI and 41,521 sheared) and 241,256 unique window IDs; the processed table collapses 11,207 overlapping source rows by retaining MspI and retains sheared-only windows. Three author-supplied intervals have end-start lengths of 402, 474, and 426 bp rather than 500 bp; because they are present in the author QC-passed S05 results and no exclusion criterion targets interval length, they were retained and exposed through window_length_bp. The raw S05 file is preserved unchanged in raw_data, as is S04, which is the run-level mSTARR library/QC metadata. The paper’s 492-window and 86% figures refer to regulatory windows overlapping the study’s tested CpG set, whereas this table contains all author-defined QC-passing mSTARR windows. The final PNAS article’s reference 89 repeats PRJNA970398 for mSTARR data, while the preprint and the dedicated BioProject identify PRJNA871297; both project links are retained in the parent metadata. No raw sequencing reads were included.

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