Dataset for "Sleep Deprivation Primes Synaptic Vulnerability Without Inducing Oxidative Damage"

This folder contains analysis scripts and metadata for the manuscript "Sleep Deprivation Primes Synaptic Vulnerability Without Inducing Oxidative Damage: A Mechanistic Reappraisal" by Lei Guo, Valentina Ferretti, and Giorgio F. Gilestro (2025). The preprint is available at https://doi.org/10.1101/2025.09.05.674430

Archive Contents

This repository provides two datasets:

1. Scripts, Metadata, and Raw Microscopy Images (guo_2025_scripts_metadata.tar.gz)    - Single compressed archive: ~3.3GB (12GB uncompressed)    - Contains all analysis scripts, experimental metadata, and confocal microscopy images

2. RNASeq Raw Data (22-part archive: guo_2025_RNASeq_part00.tar through part21.tar)    - Multi-part tar archive for easier distribution (44GB, expands to about the same)    - Contains raw FASTQ files and kallisto output (see section below)

Extracting the Scripts and Metadata Archive

To extract the complete scripts and metadata dataset:

tar -xzf guo_2025_scripts_metadata.tar.gz

Data Structure

The extracted scripts/metadata archive contains organized data and analysis scripts structured by manuscript figure:

2_Scripts_Metadata/
├── Figure_1/                  # Sleep deprivation without ROS accumulation
│   ├── 1A_Normal_food/        # Standard food conditions
│   ├── 1B_Agar_food/          # Minimal sucrose diet
│   ├── 1C_Axenic/             # Microbiome-free flies
│   └── 1D_29temp_SD/          # Higher temperature conditions
├── Figure_2/                  # Paraquat stress vs sleep deprivation
│   ├── 2BC_Paraquat/          # ROS induction and survival analysis
│   └── 2DEF_RNA_Seq/          # Transcriptomic analysis
├── Figure_3/                  # Physical and psychological stress paradigms
│   ├── 3A_Shaker_with_food/   # Mechanical stress survival
│   ├── 3BC_Physical_stress/   # ROS from mechanical perturbation
│   ├── 3DEF_Social_defeat/    # Psychological stress paradigm
│   └── 3GHI_ROS_mice/         # Mouse restraint stress validation
├── Figure_4/                  # Thermogenetic activation analysis
│   ├── AB-Sleep/              # Sleep profiles during activation
│   └── DEFG_ROS_histograms/   # ROS distribution analysis
├── Figure_5/                  # Trauma vulnerability and mutant analysis
│   ├── 5B_CantonS_SD_Survival/    # Sleep deprivation + trauma
│   ├── 5CD_Mutants/               # Short-sleep mutant responses
│   ├── 5E_dunce/                  # Synaptic plasticity mutant
│   └── 5F_CantonS-rotation/       # Control mechanical stimulation
└── ROS_RAW_IMAGES/            # Raw confocal microscopy images
    └── Fig1-Fig5/             # Organized by figure panels

Each figure folder contains:
├── Data/                      # Raw behavioral data (.pkl files)
├── Metadata/                  # Experimental metadata (.csv files)
├── Plot/                      # Generated figures (.png, .svg, .pdf)
├── Scripts/                   # Analysis notebooks (.ipynb, .R)
├── ROS/Quantifications/       # ROS fluorescence measurements
└── Sleep_profile/             # Sleep behavior analysis

RNASeq Raw Data Archive

The complete RNA-seq dataset used for Figure 2 transcriptomic analysis is provided as a multi-part tar archive. This archive contains the raw sequencing data and quantification results from the paraquat stress versus sleep deprivation comparison experiment.

Archive Contents

The dataset includes:

• Raw FASTQ files (00_fastq/): Untrimmed paired-end Illumina sequencing reads for all experimental conditions   - Control + SD (Sleep Deprivation): 3 biological replicates   - Control + no-SD: 3 biological replicates   - Paraquat + SD: 3 biological replicates   - Paraquat + no-SD: 3 biological replicates
• Kallisto output (02_output/): Transcript abundance quantification results   - abundance.h5: HDF5 format abundance estimates   - abundance.tsv: Tab-separated abundance tables   - run_info.json: Run statistics and parameters

Reconstructing the RNASeq Archive

The RNAseq data is split into 22 separate tar files (guo_2025_RNASeq_part00.tar through part21.tar), each ≤2GB for easier distribution. To reconstruct the complete dataset:

cat guo_2025_RNASeq_part*.tar | tar -xv

This will extract the complete 40-889601268/ directory containing all raw FASTQ files and analysis outputs.

Analysis Pipeline

The transcriptomic analysis was performed using:

1. Quality trimming: Trim Galore (results excluded from archive)
2. Quantification: kallisto v0.46.1 against Drosophila melanogaster transcriptome
3. Differential expression: DESeq2 analysis (scripts in Figure_2/2DEF_RNA_Seq/)

Reproducibility

All analysis scripts are provided as:

• Jupyter notebooks (Python) for behavioral and imaging data
• R scripts for transcriptomic analysis and statistical testing
• Fiji/ImageJ macros for automated ROS quantification

Data Availability

While under review, data and scripts are available at: https://lab.gilest.ro/papers/stress-not-sleep/ Upon publication, all materials will be archived in this Zenodo repository with permanent DOI.

Contact

Corresponding Author: Giorgio F. Gilestro (giorgio@gilest.ro)   First Author: Lei Guo   Department of Life Sciences, Imperial College London, UK

Funding

This work was supported by BBSRC grant BB/W016176/1 and facilities supported by Wellcome Trust (104931/Z/14/Z) and BBSRC (BB/L015129/1).