scComplete-Seq Reveals Comperehensive Picture of Transcriptome
Many of the underlying mechanisms of intercellular gene expression heterogeneity are still unknown despite the advancements in single-cell transcriptomics. One of the reasons is the partial detection of non-coding RNA types due to their small size and lack of polyadenylation. The ability to detect gene expression dynamics comprehensively would shed light on the regulation and disease-leading dysregulation in a cell-specific manner, and contribute to the discovery of biomarkers and novel therapeutics. We developed scComplete-seq, a single cell assay that enables concurrent profiling of total RNA. After successful integration of it on available high-throughput single-cell platforms, we validated it on cancer cell lines and applied it to peripheral blood mononuclear cells (PBMCs). This unveiled the distinct regulation of tRNAs, mtRNAs and enhancer RNA in monocytes, T and B cells upon Lipopolysaccharide (LPS) and PMA/Ionomycin stimulation compared to resting cells. Overall design: For scComplete-seq, 10X Chromium platform was used with modified reagents and protocol. First, additonal E. Coli PolyA polymerase, ATP and modified TSO with LNA were added to the chromium v3.1 single index kit reagent (RT Reagent B, Reducing Agent B and RT Enzyme C). After the encapsulation, cells were incubated additional 37C for 25min before the reverse transcription and template switching. The assay is benchmarked using three different cells lines mixture consisting of K562, MCF7 and HEK29T. A total of 4800 cells loaded to Chromium device after hashing the samples with hashtags. scComplete-seq is further used to discover the rewiring of coding and non-coding transcriptome in unstimulated, LPS stimulated and PMA/Ionomycin stimulated PBMCs.