Biology subjects

Womersley, H. J.

Publications and source records attributed to Womersley, H. J..

3 recordsLinked to original sources

bioRxiv2021

Time-resolved assessment of single-cell protein secretion by sequencing

Secreted proteins play critical roles in cellular communication and functional orchestration. Methods enabling concurrent measurement of cellular protein secretion, phenotypes and transcriptomes are still unavailable. Here, we describe time-resolved assessment of protein secretion from single cells by sequencing (TRAPS-seq). Released proteins are trapped onto cell surface via affinity matrices, and the captured analytes together with phenotypic markers can be probed by oligonucleotide-barcoded antibodies and simultaneously sequenced with transcriptomes. We used TRAPS-seq to interrogate secretion dynamics of pleiotropic cytokines (IFN-{gamma}, IL-2 and TNF-) of early activated human T lymphocytes, unraveling limited correlation between cytokine secretion and its transcript abundance with regard to timing and strength. We found that early central memory T cells with CD45RA expression (TCMRA) are the most effective responders in multiple cytokine secretion, and polyfunctionality involves unique yet dynamic combinations of gene signatures over time. TRAPS-seq presents a useful tool for cellular indexing of secretions, phenotypes, and transcriptomes at single-cell resolution.

immunology↗

bioRxiv2021

Heatrich-BS enables efficient CpG enrichment and highly scalable cell-free DNA methylation profiling

Genome wide analysis of cell-free DNA (cfDNA) methylation profile has been shown to be a promising approach for sensitive and specific multi-cancer detection. However, scaling these assays for clinical translation is impractical due to the high cost of whole genome bisulfite sequencing. We showed that the small fraction of GC-rich genome is highly enriched in CpG sites and disproportionately harbored the majority of cancer-specific methylation signature. Here, we report on the simple but effective Heat enrichment of CpG-rich regions for Bisulfite Sequencing (Heatrich-BS) platform that enables focused methylation profiling in these highly informative regions. Our novel method and bioinformatics algorithm enable high accuracy and sensitivity in tumor burden estimation and quantitative monitoring of colorectal patient response to treatment, at much reduced sequencing requirement. Heatrich-BS holds great potential for highly scalable screening and regular monitoring of cancer using liquid biopsy.

genomics↗

bioRxiv2020

Label-free lymphocytes reconstitution using side scatter for optimal T cell manufacturing

Lymphocyte biology research commonly involves purification of lymphocyte subpopulations by fluorescence-activated cell sorting (FACS) or immunomagnetic separation (IMS), both of which typically rely on antibody labeling of validated cell markers. Methods enabling label-free segregation of lymphocyte subpopulations would be invaluable with regard to less-perturbation, simplicity and cost-effectiveness. Here, we introduce TRuST, a label-free approach for T cell reconstitution using side-scatter (SSC). TRuST-sorted SSClow cells enrich for CD4+ T and naive T cells, while SSChigh cells enrich for CD8+ T, NK and differentiated T cells. Enrichment purity can be improved by computational gate design. SSClow cells have superior expansion capacity and generate more central memory precursors with naive-resembling cytokine responses. Moreover, we find that both T cell differentiation status and CD4/CD8 T ratio in the starting cellular material are critical attributes predicting T cell product quality and quantity. TRuST presents an effective and reliable technique for label-free lymphocytes selection and reconstitution.

immunology↗