Abstract

RNA sequencing, along with single-cell RNA sequencing, has been gaining popularity in the last decade and a half. Numerous commercial and non-commercial methods to perform transcriptome analysis exist. Despite it being so widespread, setting up an RNA sequencing platform, especially a single-cell RNA sequencing platform, is not without its own pitfalls. In this work, we showed the capabilities of our improved single-cell and bulk sequencing platform – SCRB-seq, in detecting transcriptional changes in BLaER1 cells after PRR stimulation as well as differentiation changes in CD4+ T cells after TCR activation. This study highlights the difficulties in identifying T helper sub-types in complex samples using scRNA-seq and bulk RNA-seq. Two of the main problems: overlapping cytokine production, and the continuous population seems to be an inherent property of T cells on the transcriptome level.