Experiment / E14Q879RQAAV-MPRA / in vivo MPRA

In vivo SN-MPRA 3′-UTR tiling library

In Vivo Massively Parallel Reporter Assay Reveals Sequence Determinants of mRNA Localization in Astrocytes

A focused in vivo AAV9 SN-MPRA tiled 130-nt fragments across astrocyte mRNA 3′ UTRs from Slc1a2/Glt1 isoforms, Sparc, and Hsbp1, with shuffled and positive controls. Reporter RNA was measured in cortex input, cortex TRAP, synaptoneurosome input, and PAP-TRAP fractions relative to AAV DNA.

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Organism: Mouse
Taxonomy ID: NCBITaxon:10090
Biosample: CL:0000127
Reference genome: mm10
Design focus: Region-focused
Region of interest: chr2:102777695-102790785,chr5:142903668-142903798,chr8:119348034-119348944,chr11:55394155-55395585,chr18:82584472-82584957

Perturbation & assay details

Basal / Untreated; AAV9 SN-MPRA library injected into P1 mouse cortex

Approximately 6,430 designed reporter elements were 130-nt 3′-UTR tiles shifted by 20 nt and linked to ten unique 9-nt barcodes per element. Inserts were cloned into the tdTomato 3′ UTR downstream of a GFAP promoter and packaged in AAV2/9. Targeted RNA-seq quantified AAV DNA, cortex input, cortex TRAP, SN input, and PAP-TRAP; reported contrasts are ribosome occupancy, local ribosome occupancy, localization, and local translation.

Processed data

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Column dictionary · 43 definitions

element_id: Unique element identifier from the tiling results summary.
element_class: Element category inferred from the source annotation: 130-nt UTR tile, scrambled control, or positive control.
gene: Gene or control symbol from the sequence annotation.
gene_isoform: Source gene/isoform label, such as Slc1a2_1 or Slc1a2_3.
isoform: Isoform label supplied by the source annotation.
tile_start_utr_nt: Start position of the 130-nt tile in the source transcript 3′ UTR.
chromosome: mm10 chromosome for elements with a genomic source coordinate.
genome_pos_start: Genomic start coordinate from the source annotation.
genome_pos_end: Genomic end coordinate from the source annotation.
strand: Genomic strand from the source annotation.
sequence: 130-nt reporter insert sequence, written 5′ to 3′.
sequence_length_nt: Length of the reporter insert in nucleotides.
gc_content_fraction: GC fraction reported for the sequence (0–1 scale).
predicted_structure: Dot-bracket RNA secondary-structure prediction from the source workbook.
predicted_deltaG_kcal_per_mol: Predicted RNA folding free energy in kcal/mol.
aav_mean_count: Mean source element count across the three AAV DNA input replicates.
aav_mean_cpm: Mean source element CPM across the three AAV DNA input replicates.
aav_n_replicates: Number of AAV DNA replicates with a numeric source CPM.
ctxin_mean_count: Mean source element count across reported cortex-input replicates.
ctxin_mean_cpm: Mean source element CPM across reported cortex-input replicates.
ctxin_n_replicates: Number of cortex-input replicates with a numeric source CPM.
ctxtrap_mean_count: Mean source element count across reported cortex-TRAP replicates.
ctxtrap_mean_cpm: Mean source element CPM across reported cortex-TRAP replicates.
ctxtrap_n_replicates: Number of cortex-TRAP replicates with a numeric source CPM.
snin_mean_count: Mean source element count across reported synaptoneurosome-input replicates.
snin_mean_cpm: Mean source element CPM across reported synaptoneurosome-input replicates.
snin_n_replicates: Number of synaptoneurosome-input replicates with a numeric source CPM.
sntrap_mean_count: Mean source element count across reported PAP-TRAP/synaptoneurosome-TRAP replicates.
sntrap_mean_cpm: Mean source element CPM across reported PAP-TRAP/synaptoneurosome-TRAP replicates.
sntrap_n_replicates: Number of PAP-TRAP/synaptoneurosome-TRAP replicates with a numeric source CPM.
ctxin_over_aav_mean_cpm_log2_ratio: Derived log2(mean cortex-input CPM / mean AAV DNA CPM); not the paper’s mixed-model expression statistic.
ribosome_occupancy_log2fc: Author-reported log2(Cortex TRAP / Cortex Input) effect estimate.
ribosome_occupancy_pvalue: Author-reported p-value for the ribosome-occupancy comparison.
ribosome_occupancy_fdr: Author-reported Benjamini–Hochberg FDR for ribosome occupancy.
local_ribosome_occupancy_log2fc: Author-reported log2(PAP-TRAP / SN Input) local-ribosome-occupancy effect estimate.
local_ribosome_occupancy_pvalue: Author-reported p-value for local ribosome occupancy.
local_ribosome_occupancy_fdr: Author-reported Benjamini–Hochberg FDR for local ribosome occupancy.
localization_log2fc: Author-reported log2(SN Input / Cortex Input) RNA-localization effect estimate.
localization_pvalue: Author-reported p-value for RNA localization.
localization_fdr: Author-reported Benjamini–Hochberg FDR for RNA localization.
local_translation_log2fc: Author-reported log2(PAP-TRAP / Cortex TRAP) local-translation effect estimate.
local_translation_pvalue: Author-reported p-value for local translation.
local_translation_fdr: Author-reported Benjamini–Hochberg FDR for local translation.

Quality control

Applied the paper’s QC: CPM-normalized barcode counts below 20 were excluded and an element required at least 7 sufficient barcodes. The supplemental S3 results summary contains 589 unique elements after the reported 5,818-barcode final library filter; all 589 source-summary rows with sequence annotations are retained here. Blank comparison fields are preserved when the source summary has no model result for that fraction-specific contrast; no values were imputed.

Curation notes

The paper states that putative outlier samples were evaluated with and without exclusion and were largely concordant, so the reported tiling summary includes them. A derived mean-CPM expression ratio is provided for orientation only; the inferential activity fields are the author-reported mixed-model estimates and FDRs. Coordinates are available only for genomic elements; scrambled and some control sequences have no genomic coordinate.

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