This readme file was generated on 2026-07-22 by Tim Godec

# GENERAL INFORMATION

* Title of Dataset: 

Data from publication: Functional Genomics Screening in Chlamydomonas reinhardtii Maps the Genetic Landscape of Tolerance to Paraquat and Diuron

## Author/Principal Investigator Information

* Name: Anže Županič
* ORCID: 0000-0003-3303-9086
* Institution: National Institute of Biology, Department of Biotechnology and Systems Biology
* Address: Večna pot 111, 1000 Ljubljana, Slovenia
* Email: <anze.zupanic@nib.si>

## Author/Associate or Co-investigator Information

* Name: Tim Godec
* ORCID: 0000-0002-1719-3107
* Institution: National Institute of Biology, Department of Biotechnology and Systems Biology
* Address: Večna pot 111, 1000 Ljubljana, Slovenia
* Email: <tim.godec@nib.si>

## Study information

* Date of data collection: 2021 to 2025
* Geographic location of data collection: Ljubljana, Slovenia
* Information about funding sources that supported the collection of the data:
  * Swiss National Science Foundation (SNSF) grant number 159966
  * Slovenian Research and Innovation Agency (ARIS) research grants: J4-2550, P4-0165, P1-020, P1-0143, P4-0463, P1-0207, J1-60001, L7-60161, J1-50034, J7-50043, P2-0438, IO-E015, P1-0403, and infrastructure grant IO-0004
  * U.S. National Science Foundation grant MCB-1914989
  * Howard Hughes Medical Institute (HHMI)
* Description:
  Genome-wide pooled loss-of-function screening in *Chlamydomonas reinhardtii* exposed to sub-lethal concentrations (EC20) of herbicides paraquat and diuron. The dataset contains mutant raw/normalized read counts, calculated mutant log fold-changes, high-confidence gene-phenotype associations, gene set enrichment analysis (GSEA) results, validation primer sequences, raw growth curves, structural docking metrics, and lipidomics profiles for wild-type and herbicide-tolerant mutant strains.
* Keywords: Functional genomics, Chlamydomonas reinhardtii, paraquat, diuron, ecotoxicology, P5B ATPase, lipidomics, vesicle trafficking

# SHARING/ACCESS INFORMATION

* Licenses/restrictions placed on the data: Creative Commons Attribution 4.0 International (CC BY 4.0)
* Links to publications that cite or use the data: <https://doi.org/10.1021/acs.est.5c17308>
* Links to other publicly accessible locations of the data: ACS Publications (<https://pubs.acs.org/doi/10.1021/acs.est.5c17308>)
* Links/relationships to ancillary data sets:
  * Fauser et al. (2022) *Nat. Genet.* 54(5):705-714. <https://doi.org/10.1038/s41588-022-01052-9> (Baseline growth rates dataset)
  * Li et al. (2019) *Nat. Genet.* 51(4):627-635.<https://doi.org/10.1038/s41588-019-0370-6> (C. reinhardtii mutant library description)
* Recommended citation for this dataset:
  Godec, T., Bleker, C., Stare, K., Lukan, T., Levak, V., Tušek Žnidarič, M., Betz, A., Kosjek, T., van Midden, K. P., Klemenčič, M., Trenti, F., Guella, G., Sepčić, K., Fauser, F., Patena, W., Jonikas, M. C., Kerenčič, M., Eleršek, T., Pietri, M., Rodet, T., Bren, U., Jukić, M., Lešnik, S., & Županič, A. (2026). Functional Genomics Screening in Chlamydomonas reinhardtii Maps the Genetic Landscape of Tolerance to Paraquat and Diuron. Environ. Sci. Technol. 60(22), 15570–15585. <https://doi.org/10.1021/acs.est.5c17308>

# DATA & FILE OVERVIEW

## File List

* `es5c17308_si_001.xlsx` Supplementary Table 5: PCR primer sequences used for Sanger sequencing verification of insertion junctions in mutants LMJ.RY0402.155567 and LMJ.RY0402.062151
(Supplementary Table 1): Raw and normalized barcode read counts with allele/gene mapping metadata across control, paraquat, and diuron treatment replicates.
* `es5c17308_si_002.xlsx` Supplemental table 6.5: Gene set enrichment analysis results of Flagellar and Greencut2 protein sets (Paraquat treatment)
* `es5c17308_si_003.xlsx` Supplemental table 7.5: Gene set enrichment analysis results of Flagellar and Greencut2 protein sets (Diuron treatment)
* `es5c17308_si_004.xlsx` Supplementary table 8: Raw growth data for Chlamydomonas reinhardtii wild-type (WT) and mutant strains (LMJ.RY0402.155567, Cre02.g093700; LMJ.RY0402.062151, Cre08.g373050)
* `es5c17308_si_005.xlsx` Supplementary table 4: High-confidence gene–phenotype relationships of diuron treatment
* `es5c17308_si_006.pdf` Supplementary figures (1–8), detailed supplementary methods (DNA laddering, confocal microscopy, LC-MS/MS, lipidomics, GMM statistical modeling, homology modeling & molecular docking), and Supplementary Tables 9–10 (docking scores).

* Additional related data collected that was not included in the current data package: Sanger sequencing electropherogram (.ab1) files and raw LC-MS/MS chromatograms.
* Are there multiple versions of the dataset? No

# METHODOLOGICAL INFORMATION

## Description of methods used for collection/generation of data

Pooled mutant screening was performed using >58,000 *C. reinhardtii* loss-of-function mutants grown in TAP medium under continuous light ($100\,\mu\text{mol photon}\cdot\text{m}^{-2}\cdot\text{s}^{-1}$) at 22°C. Cultures were exposed to EC20 concentrations of paraquat ($42\,\mu\text{g/L}$) or diuron ($19\,\mu\text{g/L}$) for ~72 hours (ca. 7 cell divisions). Genomic DNA was extracted via phenol/chloroform/isoamyl alcohol, and insertion cassette barcodes were amplified by PCR and sequenced on an Illumina HiSeq 2000 platform.

## Methods for processing the data

Raw sequencing reads were trimmed with Cutadapt to isolate barcode sequences and collapsed to unique reads using `fastx_collapser`. Counts were normalized to 100 million reads per library. Mutant phenotypes were computed as $log_{10}(\text{normalized treatment count} / \text{normalized control count})$. High-confidence gene-phenotype associations were determined by aggregating independent mutant alleles using Fisher's exact test (with Benjamini-Hochberg FDR correction) and complemented by a 2-step Gaussian Mixture Model (GMM) with Bayesian estimators (MAP/EAP).

## Instrument- or software-specific information needed to interpret the data

* Sequencer: Illumina HiSeq 2000
* Flow Cytometer: MACSQuant Analyzer 10
* Cell Counter: CASY Automated Cell Counter
* Confocal Microscopes: Leica Stellaris 5 and Leica Stellaris 8 (LAS AF Lite software)
* Electron Microscope: Talos L120C TEM with Ceta 16M camera (Velox software)
* LC-MS/MS: Shimadzu Nexera UHPLC connected to AB Sciex QTRAP 4500 MS/MS (Analyst v1.6.3)
* LC-Ion Trap MS: Model 1100 Series HPLC (Hewlett Packard) coupled to Esquire LC Ion Trap MS (Bruker; DataAnalysis 3.0 software)
* Software/Packages: Cutadapt, Fastx-toolkit, R (packages: `drc`, `fgsea`, `clusterProfiler`, `Pathview`), AutoDock Vina / Vinardo, YASARA, AlphaFold3.

* Standards and calibration information: LC-MS/MS matrix-matched calibration curve was established between $2.5\,\text{ng/mL}$ and $1\,\mu\text{g/mL}$ using Paraquat-$d_8$ as an internal standard ($R^2 = 0.998$).
* Environmental/experimental conditions: $22^\circ\text{C}$, continuous illumination at $100\,\mu\text{mol photons}\cdot\text{m}^{-2}\cdot\text{s}^{-1}$, liquid TAP medium, continuous shaking at 120–200 rpm.
* Describe any quality-assurance procedures performed on the data: Blank samples, process blanks, and spiked quality control samples (LOQ, medium, high) were routinely run during LC-MS/MS. Barcode sequencing of treated samles utilized biological duplicates ($R^2 = 0.94 - 0.95$). Filtering excluded alleles with 0 counts, insertions in 3' UTR, or mapping confidence $< 52\%$.
* People involved with sample collection, processing, analysis and/or submission: Tim Godec, Carissa Bleker, Katja Stare, Tjaša Lukan, Valentina Levak, Magda Tušek Žnidarič, Alexander Betz, Tina Kosjek, Katarina P. van Midden, Marina Klemenčič, Francesco Trenti, Graziano Guella, Kristina Sepčić, Friedrich Fauser, Weronika Patena, Martin C. Jonikas, Maruša Kerenčič, Tina Eleršek, Mélanie Pietri, Thomas Rodet, Urban Bren, Marko Jukić, Samo Lešnik, Anže Županič.