The advances of single-cell RNA-sequencing (scRNA-seq) allow to characterize the myeloid cell landscape providing a comprehensive map of monocyte/macrophage transitions in diseased tissue with unprecedented resolution. However, the short read length of conventional sequencing and the selective targeting of the 3" or 5" end of RNA molecules hinders the capture of full-length RNAs. Consequently, isoform elucidation remains elusive and valuable splicing information is lost. In contrast, long-read sequencing as provided by Pacific Biosciences conserves the full-length of the transcripts and detects multiple isoforms of the same gene. Here we hampered single-cell MAS-ISO-sequencing (scMAS-ISO-seq), a long-read sequencing approach based on the concatenation of cDNA fragments into long arrays [1]. We applied scMAS-ISO-seq to profile the transcriptomic landscape of murine monocyte-derived macrophages in diseased tissues. Using scMAS-ISO-seq, we unveiled cell-type-specific isoform utilization and differential splicing events among macrophage and monocyte populations. Additionally, our approach resolved differential isoform usage temporarily, correlating distinct isoforms with pseudotime ordering. We demonstrated scMAS-ISO-seq's potency in read length, throughput, and cell type identification and captured thousands of unique isoforms. scMAS-ISO-seq potentially emerges as a robust technology for investigating isoform dynamics in immunology and infection research, offering an unprecedented level of transcriptomic information.
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