{"title":"Assessing the Influence of Selected Permeabilization Methods on Lymphocyte Single-Cell Multi-Omics.","authors":"Shifan Ding, Na Lu, Hassan Abolhassani","doi":"10.3390/antib14010015","DOIUrl":null,"url":null,"abstract":"<p><p>(1) Background: Single-cell multi-omics is a powerful method for the dissection and detection of complicated immunologic functions and synapses. However, most currently available technologies merge datasets of different omics from separate portions of the same sample to generate combined multi-omics. This process is a source of bias, mainly in the field of immunology on cells originating from pluripotent hematopoietic stem cells with high flexibility during maturation. (2) Methods: Although new multi-omics approaches have been developed to use the advantages of cellular and molecular barcoding and next-generation sequencing to solve this issue, one of the main current challenges is intracellular proteomics, which should be combined with other omics data with high importance for immune system studies. We designed this study to evaluate previously recommended minimal permeabilization and fixation methods on the quality and quantity of transcriptomics and proteomics data generated by the BD Rhapsody™ Single-Cell Analysis System. (3) Results: Our findings showed that high-throughput sequencing with advanced quality and read-out is required for the combination of multi-omics outcomes from a permeabilized single cell. Therefore, the HiseqX platform was selected for further analysis. The effect of immune stimulation was observed clearly as the separated clusters of helper and cytotoxic T cells using unsupervised clustering. Importantly, fixation and permeabilization did not affect the general expression profile of unstimulated cells. However, fixation and permeabilization were proved to negatively impact the detection of the whole transcriptome for single-cell assay. Nevertheless, about 60% of the transcriptomic signature of the stimulation was detected. If the measurement of combined surface and intracellular markers is required to be achieved, the modified fixation and permeabilization method is recommended because of a lower transcriptomic loss and more precise proteomic fingerprint detected. (4) Conclusions: The findings of this study support the potential possibility for integrating intracellular proteomics, which needs to be optimized and tested with newly designed oligonucleotide-tagged antibodies targeting intracellular proteins.</p>","PeriodicalId":8188,"journal":{"name":"Antibodies","volume":"14 1","pages":""},"PeriodicalIF":3.0000,"publicationDate":"2025-02-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11843891/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Antibodies","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.3390/antib14010015","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"IMMUNOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
(1) Background: Single-cell multi-omics is a powerful method for the dissection and detection of complicated immunologic functions and synapses. However, most currently available technologies merge datasets of different omics from separate portions of the same sample to generate combined multi-omics. This process is a source of bias, mainly in the field of immunology on cells originating from pluripotent hematopoietic stem cells with high flexibility during maturation. (2) Methods: Although new multi-omics approaches have been developed to use the advantages of cellular and molecular barcoding and next-generation sequencing to solve this issue, one of the main current challenges is intracellular proteomics, which should be combined with other omics data with high importance for immune system studies. We designed this study to evaluate previously recommended minimal permeabilization and fixation methods on the quality and quantity of transcriptomics and proteomics data generated by the BD Rhapsody™ Single-Cell Analysis System. (3) Results: Our findings showed that high-throughput sequencing with advanced quality and read-out is required for the combination of multi-omics outcomes from a permeabilized single cell. Therefore, the HiseqX platform was selected for further analysis. The effect of immune stimulation was observed clearly as the separated clusters of helper and cytotoxic T cells using unsupervised clustering. Importantly, fixation and permeabilization did not affect the general expression profile of unstimulated cells. However, fixation and permeabilization were proved to negatively impact the detection of the whole transcriptome for single-cell assay. Nevertheless, about 60% of the transcriptomic signature of the stimulation was detected. If the measurement of combined surface and intracellular markers is required to be achieved, the modified fixation and permeabilization method is recommended because of a lower transcriptomic loss and more precise proteomic fingerprint detected. (4) Conclusions: The findings of this study support the potential possibility for integrating intracellular proteomics, which needs to be optimized and tested with newly designed oligonucleotide-tagged antibodies targeting intracellular proteins.
期刊介绍:
Antibodies (ISSN 2073-4468), an international, peer-reviewed open access journal which provides an advanced forum for studies related to antibodies and antigens. It publishes reviews, research articles, communications and short notes. Our aim is to encourage scientists to publish their experimental and theoretical results in as much detail as possible. There is no restriction on the length of the papers. Full experimental and/or methodical details must be provided. Electronic files or software regarding the full details of the calculation and experimental procedure - if unable to be published in a normal way - can be deposited as supplementary material. This journal covers all topics related to antibodies and antigens, topics of interest include (but are not limited to): antibody-producing cells (including B cells), antibody structure and function, antibody-antigen interactions, Fc receptors, antibody manufacturing antibody engineering, antibody therapy, immunoassays, antibody diagnosis, tissue antigens, exogenous antigens, endogenous antigens, autoantigens, monoclonal antibodies, natural antibodies, humoral immune responses, immunoregulatory molecules.