DOI: 10.5061/dryad.d2547d8h0
title: Library transgenesis in zebrafish through delayed site-specific mosaic integration for in vivo pooled screening of transgenes

description: This dataset is from a study developing a zebrafish library transgenesis
 method using delayed site-specific integration to achieve
 single-transgene-per-cell mosaicism with high library diversity. The raw
 imaging data includes confocal Z-stacks (.nd2 files) of zebrafish larvae
 expressing a 2-component library of fluorescent proteins (GFP and
 mScarlet) in neurons, captured across multiple brain regions to assess
 transgene mutual exclusivity in mosaic animals. The raw sequencing data
 (.fastq files) include Illumina amplicon reads generated from genomic DNA
 of 12 mosaic larvae with integrated libraries of barcoded GFP constructs
 (6978-R2), as well as the injected source barcode library (7462-R1).
 Larvae were injected with a complex library of barcoded plasmids, enabling
 quantification of independent integration events.
description: # Library transgenesis in zebrafish through delayed site-specific mosaic
 integration for in vivo pooled screening of transgenes Dataset DOI:
 [10.5061/dryad.d2547d8h0](https://doi.org/10.5061/dryad.d2547d8h0) ##
 Description of the data and file structure This dataset contains confocal
 microscopy images and amplicon sequencing data characterizing a zebrafish
 library transgenesis method using delayed site-specific integration. This
 dataset includes all the raw imaging data associated with Fig. 2, S1, S2,
 and Table S1, and all the raw Illumina sequencing data associated with
 Fig. 3, S3, S4, S5, S,6 and Tables S2, S3. ### Files and variables ###
 GFP-mScarlet Fluorescent Library Imaging **Format:** `.nd2` (Nikon raw
 imaging format, confocal hyperstacks)**.** **Experimental setup:** pIGLET
 embryos were injected wita h 50:50 mix of GFP-CAAX and mScarlet-CAAX
 plasmids. Larvae imaged at 3 dpf or 5 dpf, live or fixed in 4% PFA,
 mounted in 1% agarose. Spinning disk confocal microscopy (Yokogawa CSU-W1
 on Nikon Ti) with 10×/40× objectives, 488 nm (GFP) and 561 nm (mScarlet)
 channels, Z-stacks. Used to quantify integration mutual exclusivity
 (prevalence of neurons with single vs. multiple transgenes per cell).
 Associated with Fig. 2, S2, Table S1. **File naming:** **Files used for
 mutual-exclusivity quantification (Table S1):**
 `[FishID]_FP-library_[Brain_Region]_[Date_acquired].nd2` **Files used to
 produce representative images (Figure 2,
 S2):**`FP-library_[Age]_[Region]_[Date].nd2` ### Barcoded GFP Library
 Imaging **Format:** `.nd2` (Nikon raw imaging format, confocal
 hyperstacks)**.** **Experimental setup:** pIGLET embryos were injected
 with a library of barcoded GFP-CAAX plasmids (each with a unique 15-nt
 random barcode). Larvae were raised to 5 dpf and imaged live before genome
 extraction. Confocal imaging (same setup as above) with 488 nm channel to
 confirm GFP expression before selecting fish for genomic DNA extraction
 and sequencing. These 12 fish correspond to the samples in sequencing file
 6978. Associated with Fig. S1. **File naming:**
 `Fish[N]_barcoded-GFP_25_5_26_25.nd2` ### Sequencing Data Files
 **Format:** `.fastq` **Experimental setup:** 12 larvae injected with
 barcoded plasmid library at 1-cell stage were raised to 5 dpf, then
 genomic DNA was extracted from whole bodies. Junction-spanning PCR
 amplified integrated barcodes and added 5-nt sample barcodes and Illumina
 overhangs. The source library was directly amplified with primers adding
 Illumina overhangs. Sequenced on Illumina NextSeq 2000 (paired-end 2×150
 bp). Associated with: Fig. 3, S3, S4, S5, S6, Tables S2, S3. **File
 naming:** `[SampleID]_001_S[RunNumber]_R[ReadDirection]_001.fastq` *
 `6978_001_S58_R2_001.fastq` - Fish-derived amplicons (12 fish pooled,
 multiplexed) * `7462_001_S101_R1_001.fastq` - Source library amplicons ##
 Code/software The complete Python pipeline for processing the sequencing
 (.fastq) data is available at:
 [https://github.com/shaharbr/library_transgenesis](https://github.com/shaharbr/library_transgenesis) The pipeline performs demultiplexing of the 12 fish samples (using the 5-nt sample barcodes) from sample 6978, extraction of 15-nt variable barcodes (via flanking anchor sequences) from both samples, error correction (Levenshtein distance ≤1 clustering), quality filtering (≥3 reads/fish, ≥2 reads for source library), and diversity analysis. Reproduces all sequencing figures (Fig. 3D-F, S3-S6) and tables (Tables S2-S3). The raw imaging files (.nd2) can be opened with ImageJ/Fiji using the Bio-Formats plugin.