Chemical-labeling-enabled C-to-T Conversion Sequencing (CLEVER-seq) Service

Current 5-formylcytosine (5fC) mapping methods typically require bulk input DNA, averaging signals across cell populations and masking crucial heterogeneity in active demethylation processes. To address this, CD BioSciences offers the innovative chemical-labeling-enabled C-to-T conversion sequencing (CLEVER-seq) service, specifically designed for single-cell, base-resolution mapping of 5fC, empowering researchers to dissect epigenetic diversity at the cellular level.

Introduction to CLEVER-seq

5-Formylcytosine (5fC) is a key oxidative intermediate in active DNA demethylation with roles in gene regulation and development. However, its study has been constrained by the inability to profile its distribution in individual cells, limiting our understanding of its cell-type-specific functions. CLEVER-seq overcomes this barrier through a clever integration of selective chemical labeling and single-cell sequencing technologies. The core principle involves the specific chemical tagging of 5fC in fixed, permeabilized single cells, converting it into a sequencing-detectable signal. This labeled DNA is then processed through a single-cell bisulfite sequencing workflow. Through specialized bioinformatic analysis, the technique generates genome-wide 5fC maps for each individual cell, revealing heterogeneity and rare cell states invisible to bulk analysis.

Fig.1 Concept and validation of CLEVER-seq for single-cell studies. (Zhu C, et al., 2017)

Features of CLEVER-seq

CLEVER-seq is engineered to unlock single-cell 5fC biology with the following key attributes:

1. True Single-Cell Resolution: Profiles 5fC modifications in individual cells, uncovering epigenetic heterogeneity within populations.
2. Base-Specific Mapping: Identifies 5fC sites at single-nucleotide precision across the genome.
3. High Chemical Specificity: Employs selective chemical reactions to label 5fC with minimal interference from 5mC, 5hmC, or 5caC.
4. Cellular Context Preservation: Compatible with cell surface protein indexing (CITE-seq) to concurrently profile 5fC and immunophenotype.
5. Scalable Cell Throughput: Adaptable to profile from hundreds to thousands of individual cells in a single experiment.

Our Services

At CD BioSciences, we deliver an end-to-end CLEVER-seq service, transforming complex cellular samples into rich, cell-resolved epigenetic datasets. This service is ideal for researchers investigating development, tissue homeostasis, or disease where cell-to-cell variation in active demethylation is critical. We manage the entire technically demanding pipeline, enabling you to discover novel 5fC-defined cell states and dynamics.

Workflow of CLEVER-seq Service

Our CLEVER-seq service follows a meticulously coordinated, multi-stage workflow designed to preserve cellular integrity while ensuring precise 5fC detection. From initial design to final interpretation, each phase is executed by specialized teams and governed by stringent quality controls, transforming complex cellular samples into reliable, single-cell resolution epigenetic datasets.

Consultation & Experimental Design

The single-cell epigenetics experts help define key parameters, including target cell number, sample preparation strategy, necessary controls, and integration plans with other omics data, ensuring the study is statistically powered and biologically insightful.

Single-Cell Sample Preparation & Delivery

Our specialists perform cell fixation, permeabilization, and the specific chemical labeling of 5fC, or provide you with optimized protocols for preparing samples in your own lab. We conduct rigorous QC on cell concentration and viability before proceeding to library construction.

Single-Cell Library Construction

Labeled single cells are processed using industry-leading microfluidic partitioning systems for high-throughput barcoding. This is followed by bisulfite conversion and library preparation within each nanoliter-scale reaction droplet, ensuring accurate cell-of-origin identification and high molecular complexity for downstream analysis.

High-Throughput Sequencing

Constructed libraries are sequenced on Illumina platforms to achieve the deep coverage required for robust 5fC calling at the single-cell level. We perform real-time sequencing run monitoring and primary data QC to guarantee data yield and quality meet our stringent standards before advanced bioinformatic processing.

Single-Cell Bioinformatics Analysis

Our dedicated bioinformatics team employs a custom pipeline for data demultiplexing, alignment, and base-resolution 5fC calling per cell. Advanced analysis includes cell clustering based on 5fC landscapes, identification of differentially modified regions between clusters, and integration with concurrent transcriptomic or protein data to build a multi-dimensional view of cell state.

Comprehensive Reporting

You receive a detailed final report containing all processed data files, publication-ready visualizations, annotated lists of cell-type-specific 5fC markers, and a clear biological interpretation of the findings. Our scientists provide a results walkthrough to ensure you can fully leverage the insights for your research.

Our Advantages

1. Versatile Sample Compatibility: Supports diverse sample types including fresh cell suspensions, cryopreserved cells, PBMCs, cells from dissociated tissues, etc.
2. Single-Cell Epigenetic Specialization: Integrates chemical biology with single-cell sequencing for precise 5fC mapping.
3. End-to-End Technical Mastery: Manages the entire complex workflow, from sensitive cell handling to specialized bioinformatic deconvolution.
4. Actionable Heterogeneity Insights: Translates data into biological discovery, identifying how 5fC variation defines cell identity and function.
5. Rigorous Quality Control: Implements stringent QC at cell viability, library complexity, and sequencing levels to ensure data reliability.

To meet diverse research needs in 5fC analysis, CD BioSciences offers an extended portfolio of complementary technologies. Our solutions include global DNA modification quantification via LC-MS/MS and ELISA for population-level assessment, as well as targeted sequencing services such as fCAB-seq and MAB-seq for base-resolution mapping. This integrated approach enables researchers to select the optimal method based on their specific experimental requirements. If you are interested in our services, please feel free to contact us for more details and quotation information of related services.

Reference 1. Zhu C, Gao Y, Guo H, et al. Single-cell 5-formylcytosine landscapes of mammalian early embryos and ESCs at single-base resolution[J]. Cell stem cell, 2017, 20(5): 720-731. e5.