Experiment / E48CZGL5OEpisomal Plasmid MPRA

MC3T3-E1 preosteoblast episomal MPRA

Dissecting functional regulatory convergence over 160 million years of therian evolution

An episomal barcode reporter assay tested orthologous thylacine-and-wolf-accelerated regions (TWARs), ancestral and outgroup orthologs, mouse ortholog controls, and cell-line controls in MC3T3-E1 mouse preosteoblasts. The library used 10-bp sliding tiles for sequences longer than 170 bp and three biological replicate pDNA/cDNA measurements.

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Organism: Mouse
Taxonomy ID: NCBITaxon:10090
Biosample: CVCL:0409
Reference genome: mm10
Design focus: Region-focused
Region of interest: Not reported / not applicable

Perturbation & assay details

Basal / Untreated

Reporter constructs were assembled in the pMPRA1 plasmid (Addgene 49349) with 15-bp randomized barcodes and inserts up to 170 bp. Longer regions were tiled with overlapping 10-bp steps; barcode sequencing produced pDNA and cDNA counts for three MC3T3-E1 biological replicates.

Processed data

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Column dictionary · 41 definitions

sequence_id: Unique Oligo identifier from the barcode count table.
element_id: Annotated parent element name (oligo_name).
region_id: Annotated TWAR or control region grouping (twar).
species_code: Source or ancestral taxon code for the tested sequence.
element_type: Library category, such as TWAR, POPC, NCPC, or Neg.
tile_id: Species-specific annotated tile name (oligo_name_frag).
tile_number: 1-based tile order within the source sequence.
tile_start_1based: 1-based start coordinate of the tile in the source sequence.
tile_end_1based: 1-based inclusive end coordinate of the tile in the source sequence.
tile_length_bp: Length of the synthesized tile in base pairs.
source_length_bp: Length of the full source element in base pairs.
insert_sequence: Lowercase reporter insert sequence after removing the flanking cloning adapters.
barcode_count_after_pdna_qc: Number of barcode observations retained after the mean pDNA threshold; retained oligos have at least 10.
mc3t3_rep1_pDNA_raw_count: Sum of MC3T3-E1 replicate 1 pDNA counts across retained barcodes.
mc3t3_rep1_cDNA_raw_count: Sum of MC3T3-E1 replicate 1 cDNA counts across retained barcodes.
mc3t3_rep2_pDNA_raw_count: Sum of MC3T3-E1 replicate 2 pDNA counts across retained barcodes.
mc3t3_rep2_cDNA_raw_count: Sum of MC3T3-E1 replicate 2 cDNA counts across retained barcodes.
mc3t3_rep3_pDNA_raw_count: Sum of MC3T3-E1 replicate 3 pDNA counts across retained barcodes.
mc3t3_rep3_cDNA_raw_count: Sum of MC3T3-E1 replicate 3 cDNA counts across retained barcodes.
mc3t3_rep1_pDNA_mean_log2_cpm: Mean barcode-level log2 pDNA CPM for MC3T3-E1 replicate 1 after normalization.
mc3t3_rep1_cDNA_mean_log2_cpm: Mean barcode-level log2 cDNA CPM for MC3T3-E1 replicate 1 after normalization.
mc3t3_rep2_pDNA_mean_log2_cpm: Mean barcode-level log2 pDNA CPM for MC3T3-E1 replicate 2 after normalization.
mc3t3_rep2_cDNA_mean_log2_cpm: Mean barcode-level log2 cDNA CPM for MC3T3-E1 replicate 2 after normalization.
mc3t3_rep3_pDNA_mean_log2_cpm: Mean barcode-level log2 pDNA CPM for MC3T3-E1 replicate 3 after normalization.
mc3t3_rep3_cDNA_mean_log2_cpm: Mean barcode-level log2 cDNA CPM for MC3T3-E1 replicate 3 after normalization.
mc3t3_rep1_median_log2_activity: Median across retained barcodes of log2 cDNA CPM minus log2 pDNA CPM for replicate 1.
mc3t3_rep2_median_log2_activity: Median across retained barcodes of log2 cDNA CPM minus log2 pDNA CPM for replicate 2.
mc3t3_rep3_median_log2_activity: Median across retained barcodes of log2 cDNA CPM minus log2 pDNA CPM for replicate 3.
mc3t3_rep1_mean_log2_activity: Mean across retained barcodes of log2 cDNA CPM minus log2 pDNA CPM for replicate 1.
mc3t3_rep2_mean_log2_activity: Mean across retained barcodes of log2 cDNA CPM minus log2 pDNA CPM for replicate 2.
mc3t3_rep3_mean_log2_activity: Mean across retained barcodes of log2 cDNA CPM minus log2 pDNA CPM for replicate 3.
mc3t3_rep1_ttest_pvalue: One-sided one-sample t-test p-value for replicate 1 activity versus the overall replicate activity mean.
mc3t3_rep2_ttest_pvalue: One-sided one-sample t-test p-value for replicate 2 activity versus the overall replicate activity mean.
mc3t3_rep3_ttest_pvalue: One-sided one-sample t-test p-value for replicate 3 activity versus the overall replicate activity mean.
mc3t3_rep1_fdr: Benjamini-Hochberg adjusted replicate 1 activity p-value.
mc3t3_rep2_fdr: Benjamini-Hochberg adjusted replicate 2 activity p-value.
mc3t3_rep3_fdr: Benjamini-Hochberg adjusted replicate 3 activity p-value.
active_oligo: Boolean active call: FDR below 0.05 in at least one replicate and below 0.10 in at least two replicates.
most_active_tile_in_element: Tile within the annotated element with the highest mean of the three median activity values among QC-passing rows.
pdna_qc_threshold_mean_log2_cpm: Mean three-replicate log2 pDNA CPM threshold used for barcode retention.
qc_pass: Boolean indicating that the oligo had at least 10 barcode observations after pDNA QC.

Quality control

Raw barcode counts were normalized to library-size CPM and log2-transformed after adding a 0.1 pseudocount. Barcode observations were retained when the mean log2 pDNA CPM across the three replicates was at least -5.50, and oligos were retained only when at least 10 barcodes passed that filter. For each retained oligo and replicate, activity was calculated as log2 cDNA CPM minus log2 pDNA CPM, summarized by the median across barcodes, and tested with a one-sided t-test against the overall replicate activity mean; p-values were BH-adjusted within replicate. An oligo was flagged active when FDR was below 0.05 in at least one replicate and below 0.10 in at least two. The resulting table contains 18,510 QC-passing oligos, including 2,025 active oligos.

Curation notes

MC3T3-E1 is mapped to Cellosaurus CVCL_0409. The public repository contains 22,110 annotated tiles and the raw count table contains 4,786,602 barcode rows. This table retains only oligos with independent barcode observations passing the stated filters. The paper reports 2,065 active sequences; the repository R code appears to use replicate-2 pDNA in the replicate-1 activity subtraction and reattaches duplicate-sequence annotations without independent barcode observations. Here the Methods-described same-replicate pDNA/cDNA pairing is used, yielding 2,025 active measured oligo groups; inferred duplicate rows were not added.

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