Muscle-specific CUT&Tag profiling dataset of UNC-130 in Caenorhabditis elegans

Abstract

Objectives

Transcription factors can exhibit tissue-dependent chromatin-binding patterns. In Caenorhabditis elegans, genome-wide transcription factor binding profiles have primarily been generated from whole-animal samples using chromatin immunoprecipitation-based approaches, which represent aggregated signals from multiple tissues. Genome-wide datasets generated using Cleavage Under Targets and Tagmentation remain limited in this organism. UNC-130 is expressed in several tissues, but muscle-specific chromatin-binding data have not been available. In addition to providing muscle-resolved binding information, such datasets may also serve as useful references for optimizing CUT&Tag experimental and computational workflows in C. elegans.

Data description

A Cre-loxP-based conditional tagging strategy was used to epitope-label endogenous UNC-130 specifically in muscle tissue. Cleavage Under Targets and Tagmentation was performed on embryonic cells to profile genome-wide chromatin occupancy. The dataset includes raw paired-end sequencing reads, processed alignment files, peak files, normalized coverage tracks, and genome-editing construct information. All data files and associated metadata are deposited in public repositories and provide a resource for examining muscle-specific chromatin occupancy of UNC-130.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12863-026-01453-3.

Objective

Transcription factors regulate gene expression by binding to specific genomic regulatory elements and play essential roles in development and tissue differentiation [1]. In Caenorhabditis elegans, genome-wide transcription factor binding profiles have primarily been generated using chromatin immunoprecipitation followed by sequencing (ChIP-seq) [2, 3]. Most available datasets are derived from whole-animal samples, representing aggregated chromatin occupancy across multiple tissues. Although Cleavage Under Targets and Tagmentation (CUT&Tag) [4] has been increasingly adopted as an alternative chromatin profiling approach to chromatin immunoprecipitation-based methods, CUT&Tag datasets for transcription factors in C. elegans remain limited. Unlike CUT&Tag experiments in homogeneous cell populations, transcription factors in multicellular organisms such as C. elegans are often expressed in only a limited subset of cells, and due to the accessibility-dependent activity of Tn5, true binding signals may be diluted by background originating from cells that do not express the factor; datasets suitable for optimizing experimental and computational strategies under such conditions remain limited.

UNC-130 is expressed in multiple tissues in C. elegans, and whole-animal ChIP-seq datasets have been reported [5–7]. However, tissue-specific chromatin occupancy profiles of UNC-130 have not been described. Generating tissue-specific chromatin-binding data may facilitate more refined analyses of transcription factor occupancy within defined cell populations. In addition, well-characterized datasets generated using defined CUT&Tag protocols can provide useful reference data for evaluating experimental conditions and computational analysis pipelines in C. elegans.

In this study, we generated a muscle-specific CUT&Tag dataset for UNC-130 in C. elegans using a Cre-loxP-based conditional epitope-tagging strategy [8]. The resulting raw sequencing data, processed peak files, and associated metadata are publicly available and provide a resource for muscle-specific chromatin-binding analysis and for benchmarking CUT&Tag datasets in C. elegans.

Data description

To generate a muscle-specific epitope-tagged UNC-130 strain, a loxP-flanked transcriptional STOP cassette (~ 8.5 kb) followed by wScarlet::3xFLAG was inserted immediately upstream of the endogenous unc-130 stop codon using CRISPR-Cas9-mediated genome editing (Fig. S1A in Data File 1). In the unrecombined configuration, the STOP cassette separates the unc-130 coding sequence from the downstream wScarlet::3xFLAG tag, preventing expression of the tagged fusion protein.

Muscle-specific recombination was achieved by expressing Cre recombinase under the control of the muscle specific hlh-1 promoter. Cre-mediated excision of the floxed STOP cassette resulted in an in-frame fusion of wScarlet::3xFLAG to the endogenous unc-130 coding sequence in hlh-1-expressing cells, enabling tissue-specific epitope tagging of UNC-130. wScarlet fluorescence was observed in ventral body wall muscle in animals carrying both the conditional allele and the hlh-1p::Cre transgene, whereas no detectable fluorescence was observed in animals lacking Cre expression, indicating efficient Cre-mediated recombination (Fig. S1BC in Data File 1). To determine whether the conditional knock-in affected endogenous unc-130 function, we examined thrashing rate, brood size, and hatching rate. No significant differences were observed in these assays compared with control animals, indicating that the knock-in allele does not measurably impair unc-130 function (Fig. S1D-F in Data File 1). Strains, donor construct, and sgRNA sequences used for genome editing are provided in Data File 5 and Data File 6.

CUT&Tag sequencing was performed to generate genome-wide chromatin-binding profiles of muscle-specific UNC-130. Two biological replicates were generated for the conditional tagging strain and two biological replicates were generated for the corresponding control strain lacking Cre expression. Single-cell suspensions prepared from dissociated C. elegans embryos were fixed with 1% paraformaldehyde for 2 min and used for library preparation. Libraries were prepared using the Hyperactive Universal CUT&Tag Assay Kit for Illumina (Vazyme, TD901) with an anti-FLAG antibody (Sigma-Aldrich, #F1804, 1:50 dilution) targeting the epitope-tagged UNC-130 protein. Following tagmentation and PCR amplification, libraries were sequenced on a DNBSEQ-T7 platform (MGI) to generate 150 bp paired-end reads. A detailed CUT&Tag protocol is provided in Data File 7.

The dataset comprises raw sequencing reads (FASTQ files) for two replicates, including muscle-specific and control samples. Raw reads were subjected to quality control and adapter trimming using fastp (v1.0.1 [9]). Clean reads were aligned to the Caenorhabditis elegans reference genome (WBcel235) using Bowtie2 (v2.4.5 [10]) with default parameters. Only uniquely mapped reads were retained. SAM files were converted to sorted BAM files using SAMtools (v1.21 [11]). Duplicate reads were identified and removed using Picard MarkDuplicates (v3.4.0 [12]). The two muscle-specific UNC-130 CUT&Tag libraries showed high duplicate-read fractions of 97.9% and 98.0%, respectively, which are comparable to the upper range reported in a recent CUT&Tag benchmark study [13]. Duplicate-removed BAM files were used for downstream analyses. Normalized coverage tracks (bigWig) were generated using deepTools (v3.4.1 [14]). Peak calling was performed using GoPeaks (v1.0.0 [15]) with corresponding control samples to identify peaks in BED format. Sequencing and analysis summary statistics, including raw and trimmed read counts, overall alignment rates, duplicate-read fractions, numbers of called peaks, FRiP scores, and replicate-overlap peak statistics, are provided in Data File 8.

Only 830 concordant peaks shared between the two biological replicates were used for subsequent peak annotation, genomic distribution analysis, gene set enrichment analysis and motif analysis. Peaks were assigned using a custom R workflow implemented with ChIPpeakAnno(v3.44.0 [16]), based on promoter/gene-body overlaps and nearest-gene assignment when applicable (Data set 1). The genomic distribution of UNC-130 peaks were analyzed using ChIPseeker(v1.46.1 [17]). Gene set enrichment analysis of UNC-130 target genes was performed using the WormBase Gene Set Enrichment Analysis tool [18, 19]. Module scores for UNC-130 peak-associated genes were calculated using Seurat (v5.4.0 [20]) and the Packer et al. 2019 embryonic single-cell RNA-seq dataset [21], supporting preferential expression of these genes in muscle lineages. To compare UNC-130 binding profiles with published transcription factor binding datasets, reciprocal overlap proportions were calculated between UNC-130 CUT&Tag peaks and ModERN ChIP-seq peak sets [3] (Fig. S2 in Data File 2). De novo motif discovery was performed using MEME-ChIP [22] through the UseGalaxy platform [23], followed by comparison of enriched motifs against the JASPAR nematode motif [24] using Tomtom(v5.3.0 [25]) (Fig. S3 in Data File 3). Reproducible UNC-130 binding signals were detected at the known 277-bp unc-129 regulatory region [5], and near the muscle genes myo-3 and unc-54, but not near the neuronal or glial target genes nca-2, glr-1, or eat-4 [26] (Fig. S4 in Data File 4).

All raw and processed data described in this Data Note, including genome-editing construct information, sequencing reads, alignment files, peak files, and coverage tracks, are publicly available in the repositories listed in Table 1 in standard formats to facilitate reuse, reanalysis, and comparative benchmarking in C. elegans. Furthermore, the conditional epitope-tagging approach described here may be broadly applicable for generating tissue-specific chromatin-binding datasets of additional transcription factors in C. elegans. In addition to its biological relevance, this dataset may serve as a practical reference for optimizing CUT&Tag experimental conditions and computational analysis workflows in C. elegans.

Table 1.

Overview of data files/data sets

Label	Name of data file/data set	File types (file extension)	Data repository and identifier (DOI or accession number)
Data file 1	Supplemental Figure 1	Portable document format (.pdf)	FigShare (10.6084/m9.figshare.31697158.v2) [27]
Data file 2	Supplemental Figure 2	Portable document format (.pdf)	FigShare (10.6084/m9.figshare.31697158.v2) [27]
Data file 3	Supplemental Figure 3	Portable document format (.pdf)	FigShare (10.6084/m9.figshare.31697158.v2) [27]
Data file 4	Supplemental Figure 4	Portable document format (.pdf)	FigShare (10.6084/m9.figshare.31697158.v2) [27]
Data file 5	Donor vector	Genbank file format (.gbk)	FigShare (10.6084/m9.figshare.31697158.v2) [27]
Data file 6	strains-and-gRNA	Microsoft Excel spreadsheet (.xlsx)	FigShare (10.6084/m9.figshare.31697158.v2) [27]
Data file 7	Detail_cut&tag_methods	Microsoft Word document (.docx)	FigShare (10.6084/m9.figshare.31697158.v2) [27]
Data file 8	Sequencing and analysis summary statistics	Microsoft Excel spreadsheet (.xlsx)	FigShare (10.6084/m9.figshare.31697158.v2) [27]
Data set 1	Raw Data	ZIP file (.zip)	Zenodo (10.5281/zenodo.20340171) [28]
Data set 2	Raw paired-end sequencing reads for UNC-130 CUT&Tag samples (SRR38514398 and SRR38514397) and no-Cre control samples (SRR38514400 and SRR38514399)	FASTQ	NCBI Sequence Read Archive / INSDC SRA Study(https://identifiers.org/insdc.sra:SRP699499) [29]

Limitations

Like any dataset, certain limitations should be considered when planning future use. The chromatin-binding profiles described here were generated using a single muscle-specific Cre driver (hlh-1p::Cre), and additional tissue-specific drivers were not examined. As a result, the dataset reflects chromatin occupancy in the subset of cells defined by hlh-1 promoter activity rather than a comprehensive survey across all muscle subtypes or other tissues.

In addition, CUT&Tag libraries were prepared exclusively from dissociated embryonic cells. Binding profiles from intact larval and adult tissues were not assessed. Consequently, the dataset represents chromatin occupancy during embryogenesis under the experimental conditions described. The muscle-specific CUT&Tag libraries showed high duplicate-read fractions, with approximately 2% of aligned reads remaining after duplicate removal. This may be expected because CUT&Tag profiles only a small enriched fraction of the genome, but may also reflect PCR amplification bias, low input material, low target abundance, or sequencing saturation. Peak reproducibility should be interpreted with caution because GoPeaks output was not compatible with IDR analysis in this workflow, and concordant peaks were therefore defined by simple overlap between the two biological replicates. The shared peaks represented 34.33% of replicate 1 peaks and 74.77% of replicate 2 peaks, indicating asymmetric replicate overlap. These technical and analytical limitations should be considered when reusing or comparing the dataset.

For comparative analyses or benchmarking purposes, users should consider these biological, developmental, technical, and analytical constraints when interpreting differences relative to whole-animal chromatin immunoprecipitation datasets or to data generated from other tissue drivers or developmental stages.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

CUT&Tag

Cleavage Under Targets and Tagmentation

ChIP-seq

Chromatin Immunoprecipitation followed by Sequencing

Cre-loxP

Cyclization recombinase—loxP recombination system

CRISPR-Cas9

Clustered Regularly Interspaced Short Palindromic Repeats—CRISPR associated protein 9

sgRNA

Single—guide RNA

BAM

Binary Alignment/Map format

SAM

Sequence Alignment/Map format

BED

Browser Extensible Data format

GSEA

Gene Set Enrichment Analysis

Author contributions

The study was conceived and supervised by Y.L. Y.G. constructed the strains and performed the CUT&Tag experiments. Y.L. analyzed the data, wrote the manuscript, and acquired funding.

Funding

This work was supported by the National Natural Science Foundation of China Grant 32400501 (Y.L.).

Data availability

The raw sequencing reads for the UNC-130 CUT&Tag and no-Cre control sequencing runs described in this Data Note are freely available from the NCBI Sequence Read Archive under SRA Study accession SRP699499 (https://identifiers.org/insdc.sra:SRP699499) [29]. The associated data files are also publicly available: Data Files 1–8 can be accessed through figshare (https://figshare.com/) [27], whereas the processed data set (Data Set 1) is available through Zenodo (10.5281/zenodo.20340171) [28]. Please see Table 1 and references [27–29] for details and links to the data.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.