Combined HaeIII/RsaI FIREWACh lentiMPRA in E14 mouse embryonic stem cells
FIREWACh: high-throughput functional detection of transcriptional regulatory modules in mammalian cellsAccessible-chromatin (nucleosome-free region) fragments released from E14 mouse ESC nuclei with HaeIII or RsaI were cloned upstream of a minimal Fgf4 promoter-GFP lentiviral reporter, transduced at low copy, and selected by GFP FACS. The processed table contains the merged final FIREWACh interval set together with matched promoter-proximal/distal gene context and auxiliary luciferase-validation measurements where the source coordinates overlap the final set.
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
Basal / Untreated
FIREWACh used the self-inactivating FpG5 lentiviral reporter, with each NFR insert immediately upstream of a minimal Fgf4 promoter driving GFP and a hygromycin-resistance marker. HaeIII (GGCC) and RsaI (GTAC) libraries were independently transduced twice (two biological replicates per library) into E14 ESCs at MOI 7, with hygromycin selection and one to two additional FACS rounds to obtain greater than 90% GFP-positive cells. At least 10^6 GFP-positive cells were collected per sort; integrated NFRs were PCR-rescued and sequenced on Illumina MiSeq 2x150-bp runs using custom primers. Each biological replicate had three technical sequencing replicates, and the final FIREWACh interval set was formed by merging the mapped results.
Processed data
50 rows per page. Click a cell to inspect its full value.
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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 · 20 definitions
- element_id
- Package-generated identifier for the final FIREWACh interval, assigned in GEO BED order.
- chromosome
- Mouse chromosome from the GEO FIREWACh BED file.
- start_mm9_bed
- 0-based BED start coordinate on mm9 from the GEO FIREWACh file.
- end_mm9_bed
- End-exclusive BED coordinate on mm9 from the GEO FIREWACh file.
- length_bp
- Interval length in base pairs, calculated as end_mm9_bed minus start_mm9_bed.
- region_class
- Promoter-proximal or distal class from Supplementary Table 1 gene-association sheets; unassigned for final intervals absent from those sheets.
- associated_gene_id
- Source transcript/gene identifier assigned by the Supplementary Table 1 proximal or distal sheet; blank when no source association was available.
- associated_gene_name
- Source associated gene symbol/name from Supplementary Table 1; blank when no source association was available.
- tss_position_mm9
- Source annotated transcription-start-site position on mm9 from Supplementary Table 1.
- tss_distance_bp
- Signed source distance between the element and associated TSS from Supplementary Table 1.
- proximal_luciferase_validation_id
- Source FS clone identifier from the FIREWAChLucAnalysis.bed sheet in Supplementary Table 3, matched to the final interval by same-chromosome overlap.
- proximal_luciferase_esc_activity_mean
- Mean E14 ESC luciferase activity relative to the minimal Fgf4 promoter for the matched FS validation construct.
- proximal_luciferase_esc_activity_sd
- Standard deviation of the matched E14 ESC luciferase activity measurement.
- proximal_luciferase_esc_p_value_vs_minimal_promoter
- Source p-value for the matched FS construct relative to the minimal Fgf4 promoter, as reported in Supplementary Table 3.
- proximal_luciferase_esc_gt2fold
- Package-derived boolean indicating whether proximal_luciferase_esc_activity_mean is greater than 2.
- distal_luciferase_clone
- Source clone number from the Distal Elements sheet in Supplementary Table 3; semicolon-separated when multiple source clones share one final interval.
- distal_luciferase_esc_activity_mean
- Mean E14 ESC luciferase activity for a construct tested with the element in a distal reporter position, relative to the source basal reporter; semicolon-separated in the same order as distal_luciferase_clone when needed.
- distal_luciferase_esc_activity_sd
- Standard deviation of the distal-position E14 ESC luciferase activity; semicolon-separated when needed.
- distal_luciferase_3t3_activity_mean
- Mean mouse 3T3 fibroblast luciferase activity for the matched distal-position construct; semicolon-separated when needed.
- distal_luciferase_3t3_activity_sd
- Standard deviation of the matched distal-position 3T3 luciferase activity; semicolon-separated when needed.
Quality control
The authors trimmed 7 bp from the 5-prime end and 44 bp from the 3-prime end of each 2x151-bp read, aligned paired reads to mm9 with BWA default settings, and removed pairs if either read failed to map, the pair was not in proper orientation, both reads had mapping quality below 25, or neither read had a unique genomic location. Targets were called where paired reads aligned entirely within a 500-bp genomic region. Input library and FIREWACh data were merged across technical replicates, with independent biological replicates processed before final pooling. For this package, the embedded non-data header line in the GEO FIREWACh BED was excluded; all 6,364 remaining intervals had valid chromosomes, integer coordinates with end greater than start, and unique intervals, so no biological data rows were removed. FACS-positive populations were reported at greater than 90% purity, and the paper reports an estimated FIREWACh false-positive rate of 0.22 from individual luciferase validation.
Curation notes
FIREWACh is a genomically integrated, flow-sort-selected lentiMPRA rather than an allele-contrast barcode MPRA; the primary high-throughput readout is the selected set of genomic intervals, not a continuous RNA/DNA activity score. The source GEO BED has one embedded header line and 6,364 data intervals, matching the paper's reported count. Supplementary Table 1 supplies proximal/distal gene context for 6,357 intervals; seven mitochondrial intervals remain unassigned. Supplementary Table 3 validation coordinates have endpoint differences from the final BED, so they were joined by same-chromosome overlap covering at least 80% of the shorter interval: all 53 proximal validation rows and 13 distal validation rows matched, representing 12 final intervals for the distal subset because source clones 10 and 21 share one interval. Seven distal validation coordinates did not overlap the final FIREWACh set and were not forced into the processed table; the complete source sheet is retained in raw_data. No allelic variant effects or barcode-level DNA/RNA count matrix was publicly available for this study.