single-cell-characterization-calcium-influx-hiv-1-infection-using

Single-Cell Characterization of Calcium Influx and HIV-1 Infection using a Multiparameter Optofluidic Platform

HIV-1 causes a chronic infection that affects more than 37 million people worldwide. People living with human immunodeficiency virus (HIV) experience ...

DNA methylation has essential roles in transcriptional regulation, imprinting, X chromosome inactivation and other cellular processes, and aberrant CpG methylation is directly involved in the pathogenesis of human imprinting disorders and many cancers. To address the need for a quantitative and highly multiplexed bisulfite sequencing method with long read lengths for targeted CpG methylation analysis, we developed single-molecule real-time bisulfite sequencing (SMRT-BS).

Quantitative and multiplexed DNA methylation analysis using long-read single-molecule real-time bisulfite sequencing (SMRT-BS).

Hematopoietic cells are continuously replenished from progenitor cells that reside in the bone marrow. To evaluate molecular changes during this process, we analyzed the transcriptomes of freshly harvested human bone marrow progenitor (lineage-negative) and differentiated (lineage-positive) cells by single-molecule real-time (SMRT) full-length RNA-sequencing. This analysis revealed a ~5-fold higher number of transcript isoforms than previously detected and showed a distinct composition of individual transcript isoforms characteristic for bone marrow subpopulations. A detailed analysis of messenger RNA (mRNA) isoforms transcribed from the  and  loci confirmed their distinct composition. The expression of proteins predicted from the transcriptome analysis was evaluated by mass spectrometry and validated previously unknown protein isoforms predicted e.g., for . These protein isoforms distinguished the lineage negative cell population from the lineage positive cell population. Finally, transcript isoforms expressed from paralogous gene loci (e.g., , , , , and ) also distinguished cell subpopulations but were only detectable by full-length RNA sequencing. Thus, qualitatively distinct transcript isoforms from individual genomic loci separate bone marrow cell subpopulations indicating complex transcriptional regulation and protein isoform generation during hematopoiesis.

Article	Year
Quantitative and multiplexed DNA methylation analysis using long-read single-molecule real-time bisulfite sequencing (SMRT-BS). BMC genomics\| PubMed ID: 25943404	2015
Single-Molecule Real-Time (SMRT) Full-Length RNA-Sequencing Reveals Novel and Distinct mRNA Isoforms in Human Bone Marrow Cell Subpopulations. Genes\| PubMed ID: 30934798	2019

Robert P. Sebra

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