Experiment / E4E3WI0NYTargeted / Cap-STARR-seq

Capture STARR-seq MPRA of pig fat-related enhancer SNPs in 3T3-L1 cells

Constructing eRNA-mediated gene regulatory networks to explore the genetic basis of muscle and fat-relevant traits in pigs

A capture STARR-seq library of 22 pig enhancer regions containing 107 population-differentiated SNP candidates was cloned into the hSTARR-seq_ORI episomal self-transcribing reporter and transfected into mouse 3T3-L1 fibroblasts. Pooled pig DNA from eight eastern and eight western pigs supplied the alleles; input plasmid DNA and 24-hour reporter RNA were sequenced, and allele-specific enhancer activity was quantified as the output/input change in alternate-allele fraction.

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Organism: Mouse
Taxonomy ID: NCBITaxon:10090
Biosample: CVCL:0123
Reference genome: susScr11
Design focus: Variant-focused
Region of interest: Not reported / not applicable

Perturbation & assay details

Basal 3T3-L1 culture; reporter RNA harvested 24 h after transfection

Pig enhancer fragments were PCR-amplified from an equal pooled DNA mixture of eight eastern and eight western pigs, sonicated to 300–400 bp, and inserted into hSTARR-seq_ORI (Addgene #99296). The pooled library was transfected into 3T3-L1 cells with Lipofectamine/jetPRIME. One input plasmid NGS library and four output RNA NGS libraries (two per biological replicate) were generated and sequenced PE150 on NovaSeq 6000. The publication merged the two biological replicates after QC because they were highly correlated. The library sequence source is pig and coordinates follow susScr11, while the assayed cellular context is mouse 3T3-L1.

Processed data

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Column dictionary · 25 definitions

variant_id: Stable variant identifier formed from chromosome, 1-based position, reference allele, and alternate allele.
chromosome: Pig chromosome as reported in Table S11.
position: 1-based pig genomic position as reported in Table S11.
reference_allele: Reference allele used for the allele-count comparison.
alternate_allele: Alternate allele used for the allele-count comparison.
target_enhancer_ids: One or more numbered capture-library enhancer targets from Table S2 that contain the variant.
target_enhancer_regions: susScr11 coordinate interval(s) of the capture-library enhancer target(s) from Table S2.
input_ref_count: Input plasmid DNA reads carrying the reference allele.
input_alt_count: Input plasmid DNA reads carrying the alternate allele.
input_other_count: Input plasmid DNA reads carrying bases other than the reported reference or alternate allele.
input_total_count: Total input plasmid DNA reads reported for the SNP.
input_alt_fraction: Derived alternate-allele fraction in the input plasmid library.
output_ref_count: Reporter RNA reads carrying the reference allele.
output_alt_count: Reporter RNA reads carrying the alternate allele.
output_other_count: Reporter RNA reads carrying bases other than the reported reference or alternate allele.
output_total_count: Total reporter RNA reads reported for the SNP.
output_alt_fraction: Derived alternate-allele fraction in the reporter RNA output.
activity_fold_change_output_over_input: Authors' output/input fold change in alternate-allele fraction; values above 1 indicate relative alternate-allele enrichment in reporter RNA.
p_value: Two-tailed Fisher's exact-test p-value reported by the authors.
fdr: False-discovery-rate-adjusted p-value reported by the authors.
reference_allele_frequency_eastern: Reference-allele frequency in the eastern pig population from Table S10.
reference_allele_frequency_western: Reference-allele frequency in the western pig population from Table S10.
allele_frequency_difference: Population allele-frequency difference reported in Table S10.
pooled_allele_frequency: Allele frequency in the pooled eastern/western datasets as reported in Table S10; the workbook does not separately label the allele for this field.
qc_pass: TRUE for rows passing the author coverage/significance criteria and package integrity/join checks.

Quality control

The authors performed read QC, PCR-duplicate removal, replicate-correlation assessment, and merged the two biological replicates for calling regulatory SNPs. Effect sizes were calculated only for SNPs with coverage greater than 20 in both input and output libraries, and significance was assessed with a two-tailed Fisher's exact test. For this package, Table S11 rows were additionally required to have valid single-nucleotide ref/alt alleles, ref and alt labels matching the base-count columns, base counts summing to the reported coverage in both libraries, coverage greater than 20 in both libraries, a matching candidate record in Table S10, an overlapping target interval in Table S2, and FDR < 0.05. All 16 retained rows pass these checks; the public result workbook reports significant SNPs only, so the 91 candidate SNPs without released effect rows are not represented in table.csv.

Curation notes

The experiment is capture STARR-seq rather than a barcode-count MPRA: each enhancer fragment self-transcribes its insert sequence, and the allelic readout is the alternate-allele fraction in output RNA relative to input plasmid DNA. The public S11 workbook contains 16 significant SNPs, matching the paper's reported 16 regulatory SNPs; S10 contains the broader 107-SNP selection list but no released effect statistics for the other 91 candidates. Target intervals are assigned by coordinate overlap with the 22 primer-defined regions, so variants in overlapping target intervals can have multiple target_enhancer_ids. No rs identifiers were provided. The source paper reports raw STARR-seq reads under GSA CRA011292/PRJCA017321, but they were omitted per package instructions.