Experiment / E58EVSI6K5' UTR / Translation Efficiency MPRA (MPTA)

Multi-frame NaP-TRAP pilot in zebrafish embryos at 6 hpf

NaP-TRAP reveals the regulatory grammar in 5’UTR-mediated translation regulation during zebrafish development

A 128-reporter synthetic 5'UTR library encoded combinations of no ORFs, uORFs, and overlapping ORFs in three reading frames. The 3xFLAG-HA-MYC reporter was injected into single-cell zebrafish embryos and FLAG, HA, and MYC NaP-TRAP pulldowns were measured at 6 hpf.

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Organism: Zebrafish
Taxonomy ID: NCBITaxon:7955
Biosample: UBERON:0000922
Reference genome: Not reported / not applicable
Design focus: Synthetic / Motif-focused
Region of interest: Not reported / not applicable

Perturbation & assay details

Basal / Untreated

The common reporter encoded FLAG, HA, and MYC epitope tags in frames 1, 2, and 3. Each replicate was split into tag-specific immunoprecipitations; all three pulldowns were normalized to the shared input with five spike-in reporters, preserving frame-specific translation in one table.

Processed data

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Column dictionary · 46 definitions

reporter_id: Synthetic multi-frame reporter identifier.
insert_sequence: Synthetic 5'UTR insert containing designed ORF combinations.
sequence_class: Synthetic multi-frame ORF-design label.
input_B1: UMI-deduplicated shared input count for biological replicate B1.
input_B2: UMI-deduplicated shared input count for biological replicate B2.
input_scale_B1: Sum of all five spike-in counts used to normalize B1 input.
input_scale_B2: Sum of all five spike-in counts used to normalize B2 input.
norm_input_B1: B1 input count divided by input_scale_B1.
norm_input_B2: B2 input count divided by input_scale_B2.
flag_pulldown_B1: UMI-deduplicated FLAG pulldown count in B1.
flag_scale_B1: Sum of all five spike-in counts used to normalize FLAG B1 pulldown.
flag_norm_pulldown_B1: FLAG B1 pulldown count divided by flag_scale_B1.
flag_translation_B1: FLAG B1 translation ratio, flag_norm_pulldown_B1 / norm_input_B1.
flag_pulldown_B2: UMI-deduplicated FLAG pulldown count in B2.
flag_scale_B2: Sum of all five spike-in counts used to normalize FLAG B2 pulldown.
flag_norm_pulldown_B2: FLAG B2 pulldown count divided by flag_scale_B2.
flag_translation_B2: FLAG B2 translation ratio, flag_norm_pulldown_B2 / norm_input_B2.
mean_flag_translation: Arithmetic mean of FLAG translation ratios across B1 and B2.
sd_flag_translation: Sample standard deviation of FLAG translation ratios.
log2_mean_flag_translation: Base-2 logarithm of mean_flag_translation.
ha_pulldown_B1: UMI-deduplicated HA pulldown count in B1.
ha_scale_B1: Sum of all five spike-in counts used to normalize HA B1 pulldown.
ha_norm_pulldown_B1: HA B1 pulldown count divided by ha_scale_B1.
ha_translation_B1: HA B1 translation ratio, ha_norm_pulldown_B1 / norm_input_B1.
ha_pulldown_B2: UMI-deduplicated HA pulldown count in B2.
ha_scale_B2: Sum of all five spike-in counts used to normalize HA B2 pulldown.
ha_norm_pulldown_B2: HA B2 pulldown count divided by ha_scale_B2.
ha_translation_B2: HA B2 translation ratio, ha_norm_pulldown_B2 / norm_input_B2.
mean_ha_translation: Arithmetic mean of HA translation ratios across B1 and B2.
sd_ha_translation: Sample standard deviation of HA translation ratios.
log2_mean_ha_translation: Base-2 logarithm of mean_ha_translation.
myc_pulldown_B1: UMI-deduplicated MYC pulldown count in B1.
myc_scale_B1: Sum of all five spike-in counts used to normalize MYC B1 pulldown.
myc_norm_pulldown_B1: MYC B1 pulldown count divided by myc_scale_B1.
myc_translation_B1: MYC B1 translation ratio, myc_norm_pulldown_B1 / norm_input_B1.
myc_pulldown_B2: UMI-deduplicated MYC pulldown count in B2.
myc_scale_B2: Sum of all five spike-in counts used to normalize MYC B2 pulldown.
myc_norm_pulldown_B2: MYC B2 pulldown count divided by myc_scale_B2.
myc_translation_B2: MYC B2 translation ratio, myc_norm_pulldown_B2 / norm_input_B2.
mean_myc_translation: Arithmetic mean of MYC translation ratios across B1 and B2.
sd_myc_translation: Sample standard deviation of MYC translation ratios.
log2_mean_myc_translation: Base-2 logarithm of mean_myc_translation.
input_total: Sum of shared input counts across B1 and B2.
qc_input_minimum: Lower of the two replicate input counts; at least 100 by construction.
n_replicates: Number of input/pulldown replicate pairs contributing to each channel mean (2).
qc_pass: True for rows retained after both input-read QC filters.

Quality control

UMI-deduplicated count tables were matched to the multi-frame reporter FASTA and normalized to all five spike-ins. Reporters with at least 100 unique input reads in both B1 and B2 were retained (98 reporters).

Curation notes

FLAG, HA, and MYC channels are intentionally kept together because they are three frame-specific readouts from the same multi-frame MPRA experiment.

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