Experiment / E2AR82557Targeted Genomic Integration MPRA

twi:CD2+ / GFP+ embryonic mesoderm eFS

Highly parallel assays of tissue-specific enhancers in whole Drosophila embryos

The pooled cCRM library was assayed in stage 11–12 Drosophila embryos carrying the twi:CD2 mesoderm marker, and GFP-positive CD2-positive cells were collected. Activity was quantified against matched twi:CD2+ input cells in three target and three input replicates.

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Organism: Fruit fly
Taxonomy ID: NCBITaxon:7227
Biosample: UNMAPPED:embryonic_mesoderm
Reference genome: dm3
Design focus: Region-focused
Region of interest: Not reported / not applicable

Perturbation & assay details

Basal / Untreated

Each approximately 1-kb cCRM was cloned upstream of a nuclear EGFP reporter driven by an Hsp70 minimal promoter in pEFS-Dest and integrated one per transformant line by phiC31 into the attP40 landing site. Stage 11–12 embryo cells were stained with Alexa647 anti-rat CD2, separated by CD2/GFP FACS gates, and the cCRM inserts were recovered from genomic DNA by nested PCR and sequenced as 50-base single-end Illumina HiSeq 2000 reads mapped with segemehl. This is a region-focused genomic integration assay with no molecular barcodes or allele pairs. For this contrast, the target samples were twi-CD2+/GFP+ rep. 1–3 and the input reference samples were twi-CD2+ input rep. 1–3.

Processed data

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Column dictionary · 33 definitions

element_id: Unique cCRM/library element identifier from the authors' Supplementary Tables 1 and 4.
chromosome: Drosophila chromosome reported for the cCRM in the dm3 coordinate string.
start: One-based genomic start coordinate reported by the authors.
end: Genomic end coordinate reported by the authors.
coordinate_span_bp: Coordinate-derived span in base pairs, calculated from the reported start and end.
sequence: Candidate cis-regulatory module sequence supplied in Supplementary Table 1.
sequence_length: Number of bases in the supplied cCRM sequence.
gc_fraction: Fraction of sequence bases that are G or C.
forward_primer: Forward primer sequence supplied for recovery/amplification of the cCRM.
reverse_primer: Reverse primer sequence supplied for recovery/amplification of the cCRM.
library_source_category: Library/source category reported for the cCRM.
input_count_rep1: Raw overlap-weighted mapped cCRM Count from input sample replicate 1; the exact sample title is documented in agent_comments and raw_data/E-GEOD-41503.sdrf.txt.
input_count_rep2: Raw overlap-weighted mapped cCRM Count from input sample replicate 2; the exact sample title is documented in agent_comments and raw_data/E-GEOD-41503.sdrf.txt.
input_count_rep3: Raw overlap-weighted mapped cCRM Count from input sample replicate 3; the exact sample title is documented in agent_comments and raw_data/E-GEOD-41503.sdrf.txt.
target_count_rep1: Raw overlap-weighted mapped cCRM Count from target sample replicate 1; the exact sample title is documented in agent_comments and raw_data/E-GEOD-41503.sdrf.txt.
target_count_rep2: Raw overlap-weighted mapped cCRM Count from target sample replicate 2; the exact sample title is documented in agent_comments and raw_data/E-GEOD-41503.sdrf.txt.
target_count_rep3: Raw overlap-weighted mapped cCRM Count from target sample replicate 3; the exact sample title is documented in agent_comments and raw_data/E-GEOD-41503.sdrf.txt.
input_count_mean: Arithmetic mean of the raw overlap-weighted mapped cCRM Count values across input replicates.
target_count_mean: Arithmetic mean of the raw overlap-weighted mapped cCRM Count values across target replicates.
input_cpm_mean: Mean input abundance after normalizing each input file's cCRM Count values to counts per million.
target_cpm_mean: Mean target abundance after normalizing each target file's cCRM Count values to counts per million.
normalized_target_input_ratio: Recomputed ratio of mean target CPM to mean input CPM; this is not the authors' DESeq statistic.
normalized_log2_enrichment: Base-2 logarithm of normalized_target_input_ratio; zero target is represented as -Inf and zero input as Inf.
input_detected_replicates: Number of input replicates meeting the paper's detection thresholds: both ends detected, at least 5 center positions covered, and at least 10 total reads.
target_detected_replicates: Number of target replicates meeting the paper's detection thresholds: both ends detected, at least 5 center positions covered, and at least 10 total reads.
author_input_abundance: Input abundance reported by the authors in Supplementary Table 4 for this contrast.
author_log2_fold_change: Authors' DESeq log2 fold change for target versus input; retained as reported.
author_padj: Authors' DESeq multiple-testing-adjusted P value for the target-versus-input contrast.
author_activity_call: Packaging call: active when author_padj is below 0.1, otherwise not_significant; blank when no author adjusted P value is available.
redfly_overlap_bp: Number of base pairs overlapping a RedFly annotated regulatory region, as reported by the authors.
hot_region_overlap: Whether the cCRM overlaps a reported hot region, as reported by the authors.
validation_call: Summary validation call reported by the authors.
validation_finding: Validation result or explanatory finding reported by the authors.

Quality control

The authors required at least 1 read from each cCRM end, at least 5 center positions covered, and at least 10 total reads; matched random genomic windows gave a cCRM-detection FDR below 5×10−5. cCRMs not detected in any input sample replicate were filtered before DESeq analysis, and the authors used DESeq size-factor estimation with adjusted P <0.1 as the activity threshold. For packaging, rows were retained when the corresponding Supplementary Table 4 inputAbundance was numeric and the Supplementary Table 1 record had a valid sequence and coordinate. Across the deposited samples, 92.50–98.59% of filtered reads were assigned to cCRMs. Inactive and depleted elements were retained; zero-target results are represented as -Inf.

Curation notes

This is one of six child contrasts from the same eFS study/library, not an allele-specific variant experiment. Target sample titles: twi-CD2+/GFP+ rep. 1–3; input sample titles: twi-CD2+ input rep. 1–3. The raw title-to-file and GEO/BioStudies sample metadata are retained in raw_data/E-GEOD-41503.sdrf.txt, and the mapped cCRM Count files are in raw_data/geo_processed/. The normalized_* columns are recomputed from each raw file's Count divided by that file's total mapped cCRM Count and are not replacements for the authors' DESeq statistics; use author_log2_fold_change and author_padj for the published contrast. Source NA/None annotations are empty CSV fields, while author -Inf/Inf values are retained. The source coordinate span and sequence length differ by one base for some cCRMs; both supplied sequence and coordinate values are preserved. The target population is GFP+CD2+ cells from twi:CD2 embryos (whole embryonic mesoderm); the input population is matched twi:CD2+ mock-sorted cells.

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