DNA methylation is a fundamental epigenetic modification that regulates gene expression, maintains genomic integrity and defines cellular identity. Conventional bulk sequencing methods, such as reduced-representation bisulfite sequencing (RRBS), have elucidated genome-wide methylation landscapes but do not reveal cell-to-cell variation, limiting insights into methylation heterogeneity during development, differentiation and disease. Here, we present multiplexed single-cell RRBS (Msc-RRBS), a streamlined and scalable protocol for parallel DNA methylation profiling of hundreds of individual cells. All enzymatic reactions, including cell lysis, restriction digestion, end repair and adapter ligation, are carried out in a single microwell, which eliminates the need for buffer exchange and minimizes DNA loss. Short, indexed adapters are used to efficiently remove adapter dimers and enable effective purification of library DNA by magnetic bead clean-up. The workflow supports the generation of sequencing libraries from single cells, pooled cells or sub-nanogram amounts of purified genomic DNA. The full workflow, from cell sorting to sequencing-ready libraries and data analysis, requires ~6 d for one or more 96-well plates. Msc-RRBS thus provides an efficient, reproducible and high-throughput approach for dissecting cell-to-cell DNA methylation heterogeneity across diverse biological and disease contexts. The experiments require basic molecular biology skills, and primary data processing can be performed by someone with limited bioinformatics training. More advanced analyses need additional computational expertise. This protocol outlines multiplexed single-cell reduced-representation bisulfite sequencing (Msc-RRBS), a streamlined and scalable approach for parallel DNA methylation profiling of hundreds of individual cells.
The dynamic structural reconstruction of Cu-based catalysts profoundly influences activity, selectivity and stability during electrochemical CO2 reduction (CO2RR). However, it lacks standardized methodologies for probing and actively managing these transformations. Here we present a modular, reproducible Protocol that integrates electrochemical control, in situ/operando spectroscopy and mechanistic analysis to systematically investigate and regulate Cu catalyst reconstruction under CO2RR conditions. This Protocol establishes a comprehensive ‘reconstruction–understanding–intervention’ workflow comprising (1) the identification and taxonomy of reconstruction phenomena, (2) quantification of electrochemical and environmental factors governing reconstruction, (3) correlation of structural dynamics with catalytic behavior through complementary in situ/operando Raman, infrared, X-ray absorption spectroscopy and quasi-in situ X-ray photoelectron spectroscopy measurements, and (4) development of rational reconstruction-control strategies that steer catalysts toward desired active states. A systematic approach is delineated to correlate reconstruction dynamics with catalytic performance and actively direct the process toward desired active states via three primary strategies: catalyst structure modulation, electrochemical operation regulation and reaction microenvironment management. The methodology is validated using representative Cu-based systems, including commercial Cu, oxide-derived Cu and bimetallic Cu–X catalysts, demonstrating enhanced performance and stability of CO2RR. This workflow also provides an adaptable framework for investigating dynamic surface evolution in other electrocatalytic reactions. This Protocol provides useful framework for the rational development of adaptive, stable and selective electrocatalysts. Copper-based catalysts undergo structural changes during the course of the chemical reaction. This protocol describes how to use in situ/operando analysis using Raman, IR, XPS and XRD to understand Cu reconstruction during CO2 electroreduction.
Mammalian embryonic development is a highly orchestrated process initiated by the fusion of the oocyte with sperm to generate the zygote. In humans, the zygote remains transcriptionally quiescent until the major wave of zygotic genome activation (ZGA) occurs around the eight-cell (8C) stage (day 3 after fertilization). These cells and the derived morula cells are totipotent: they have the capacity to form a whole individual. Our understanding of human totipotency is very limited because of ethical concerns using embryos and the scarcity of material available for research. Recently, we established a controllable transgene-free methodology to generate totipotent eight-cell embryo-like cells (8CLCs) from human pluripotent stem cells (PSCs) in vitro. These 8CLCs are produced using a novel medium, formulated by us, containing specific chemical compounds and cytokines. Here, we provide a detailed protocol for inducing, isolating and characterizing 8CLCs generated with this medium. The induction process can be done either in a stepwise manner (primed-naive-8CLC) that requires only 5 d starting from naive PSCs or directly from primed PSCs, which takes ~7 d. The resulting 8CLCs exhibit transcriptional and epigenetic features resembling those of human 8C embryo cells. On the basis of our experience, we expect that an individual with ~1 year of experience working with human PSC culture would be able to carry out this protocol. Our approach provides a valuable model for studying human early embryogenesis, particularly the molecular events surrounding ZGA. This protocol describes the generation of eight-cell embryo-like cells (8CLCs) from human pluripotent stem cells. The resulting 8CLCs exhibit transcriptional and epigenetic features resembling those of human 8C embryo cells.
The engineering of living cells represents a promising biomedical frontier that enables the design of cells with tailored functionalities for advanced therapeutic applications. Genetic manipulation serves as a primary approach in cell engineering, yet it faces inherent limitations, including the complexity of multigene editing and poor cross-species applicability, which restrict the development of cells with sophisticated functionalities. Therefore, flexible and versatile engineering strategies capable of functionalizing living cells to address diverse therapeutic requirements are highly desirable. Given its pivotal role in mediating cellular interactions, the cell surface is an attractive target for directing cell engineering. The diverse functional groups present in surface biomolecules offer abundant chemical modification sites, making them highly amenable to functionalization. Leveraging this inherent chemical accessibility, we have recently developed a flexible and versatile platform for surface functionalization of living cells through in situ dopamine polymerization that allows us to design personalized living cells with customizable functions by tuning the surface components. Here we provide a detailed protocol describing two distinct methods for bacterial functionalization. The first method uses dopamine polymerization-mediated mono-functionalization to construct mucus-penetrating bacteria that can reinforce intestinal mucosal barrier to prevent colitis. The second method uses dopamine polymerization-mediated dual-functionalization to generate synergy-immunoactivation bacteria that can simultaneously induce anticancer and antiviral immunity to treat cancer and prevent infection. Excluding bacterial culture, preparation of mucus-penetrating bacteria and synergy-immunoactivation bacteria takes ~3 h and 1 h, respectively. We anticipate that this protocol can offer valuable guidance for the engineering of living cells with designable and tailorable functionalities for innovative cell-based therapy.
The human cerebrovasculature is a critical yet historically understudied component of neurological health. Dysfunction of the diverse endothelial, mural, and perivascular cells that comprise cerebral vessels is central to diseases ranging from stroke to Alzheimer's disease. However, characterizing these cell populations at a molecular level has proven exceptionally challenging. Encased within a robust basement membrane, vascular cells resist standard dissociation methods, leading to their systematic depletion and underrepresentation in existing single-nucleus genomic atlases. This has created a major blind spot in neuroscience. To overcome this barrier, we developed vessel isolation and nucleus extraction for sequencing (VINE-seq) and its advanced iteration, MultiVINE-seq. The protocol provides a robust, reproducible workflow for the enrichment and high-resolution profiling of vascular, perivascular, and immune cells from fresh or frozen human and mouse brain tissue. First, intact vessels (predominantly capillaries and small arterioles/venules, 100 µm in diameter) are isolated from homogenized brain tissue via dextran-based density-gradient centrifugation, separating the vascular pellet from myelin and the parenchymal fraction. Second, the collected vessels are rigorously washed over a cell strainer to remove trapped contaminants. A critical innovation lies in the third stage: the optimized extraction of nuclei from purified vessels using enzymatic digestion. After extraction, the protocol uses fluorescence-activated cell sorting (FACS) to ensure collection of high-purity nuclei suitable for widely used droplet-based sequencing platforms (e.g., 10x Genomics single cell 3' or multiome). This protocol requires 4-5 h to complete and can be carried out by researchers with single-cell and flow cytometry training.
