Experiment / E2RYXGK44AAV-MPRA / in vivo MPRA

In vivo AAV9 barcoded promoter library screen across 16 mouse tissues

Identification of a robust promoter in mouse and human hepatocytes by in vivo biopanning of a barcoded AAV library

A pooled library of 53 distinct promoter or enhancer-promoter constructs, each linked to a unique 15-nt barcode in the 3′ UTR of an EYFP reporter, was individually packaged in AAV9 and injected intravenously into six female C57BL/6 mice. Barcode sequencing of tissue gDNA and cDNA from 16 tissues was used to estimate promoter biodistribution and transcriptional activity.

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

Perturbation & assay details

Pooled AAV9 promoter library; 1 × 10^11 vector genomes per promoter construct per mouse by tail-vein injection; 2-week endpoint

Single-stranded AAV9 vectors carried promoter-EYFP-bovine-growth-hormone-polyadenylation cassettes with promoter-specific 15-nt barcodes in the EYFP 3′ UTR. Six female C57BL/6 mice received an equal pooled library; brain, diaphragm, eye, fat, gut, heart, kidney, liver, lung, lymph nodes, skeletal muscle, ovary, pancreas, skin, spleen, and stomach were harvested. Amplicon sequencing quantified barcode fractions in genomic DNA (vector biodistribution) and cDNA (transcriptional output).

Processed data

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Column dictionary · 27 definitions

element_id: Stable generated identifier for the promoter element.
promoter_name: Promoter name from Table S1; minor barcode-label aliases were resolved to the Table S1 spelling.
barcode: 15-nt barcode assigned to the promoter in the primary in vivo library.
promoter_sequence: Promoter sequence from supplementary Table S1.
promoter_length_bp: Promoter length in base pairs from Table S1.
supposed_specificity: Reported intended specificity class or description from Table S1.
origin: Reported sequence origin from Table S1.
tissue: Mouse tissue abbreviation used by the study for the sampled tissue.
n_replicates: Total biological mouse replicates planned for the tissue (6).
n_qc_replicates: Number of paired mouse gDNA/cDNA observations retained after promoter-level QC.
dna_count_mean: Mean mapped barcode read count in gDNA across retained mice; counts are from the per-run FASTQ prefixes.
dna_count_sd: Population standard deviation of mapped gDNA barcode counts across retained mice.
cdna_count_mean: Mean mapped barcode read count in cDNA across retained mice; counts are from the per-run FASTQ prefixes.
cdna_count_sd: Population standard deviation of mapped cDNA barcode counts across retained mice.
dna_fraction_mean: Mean fraction of all known primary-library barcode reads represented by this barcode in gDNA.
dna_fraction_sd: Population standard deviation of the gDNA barcode fraction across retained mice.
cdna_fraction_mean: Mean fraction of all known primary-library barcode reads represented by this barcode in cDNA.
cdna_fraction_sd: Population standard deviation of the cDNA barcode fraction across retained mice.
qPCR_bulk_expression_mean: Mean tissue-level EYFP/housekeeper bulk expression normalization factor Cβ from supplementary Table S3.
normalized_expression_mean: Mean promoter activity Rαβ, calculated as (cDNA barcode fraction / gDNA barcode fraction) × Cβ.
normalized_expression_sd: Population standard deviation of Rαβ across retained mice.
efficiency_score_mean: Mean tissue-normalized promoter efficiency score Eαβ = 100 × Rαβ divided by the sum of promoter R values in that tissue.
efficiency_score_sd: Population standard deviation of the promoter efficiency score across retained mice.
specificity_score_mean: Mean promoter-normalized tissue specificity score Sαβ = 100 × Rαβ divided by the sum of tissue R values for that promoter.
specificity_score_sd: Population standard deviation of the tissue specificity score across retained mice.
log2_normalized_expression_mean: Base-2 logarithm of normalized_expression_mean.
qc_pass: Whether the promoter-by-tissue summary passed the paired-observation QC rule (at least four retained mice).

Quality control

The package extracted DNA-only reads containing the documented forward barcode structure (ATGCTC + 15-nt barcode + CAGGGT) at the expected read position and restricted matches to the 53 primary-library barcodes in Table S3. Because the 192 source FASTQ archives total approximately 84 GB, barcode counts were calculated from the first 20 MiB of each run; run-level depth and mapping statistics are retained in raw_data/qc_summary.tsv. All 192 runs passed package-level QC (at least 100,000 prefix reads, at least 100,000 known-barcode reads, and at least 47 of 53 barcodes observed). For promoter-by-tissue summaries, paired gDNA/cDNA mouse observations with gDNA barcode counts below 10 were excluded; three observations were excluded, while zero cDNA counts were retained. Summary rows require at least four paired QC mice.

Curation notes

This experiment packages the primary six-mouse in vivo AAV9 screen deposited under BioProject PRJNA1209753: 192 runs covering 6 mice × 16 tissues × gDNA/cDNA. The paper also describes AAV-DJ in vitro mini-libraries, primary human hepatocytes, and a liver-humanized FRGN mouse validation; those validation assays were not assigned a second experiment because a complete, separately deposited barcode count matrix was not available in the source package. Counts in the raw and processed tables are high-depth prefix estimates rather than full-archive recounts, and this is documented in raw_data/source_manifest.txt. Table S1 sequences and Table S3 normalization factors are carried through directly; normalized expression, efficiency, and specificity summaries are calculated from the extracted barcode counts.

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