Nascent RNA sequencing of peripheral blood leukocytes reveal gene expression diversity

Nuclear Run-On sequencing is a powerful method to measure transcription with high resolution, sensitivity, and directional information, which provides alternative perspective from existing methods such as chromatin immunoprecipitation or mRNA sequencing. Current form of Nuclear Run-On assays such as Precision Run-On sequencing (PRO-seq) involves multiple RNA chemistry steps including RNA end repairs and ligations. These have limited the widespread use of PRO-seq by requiring robust RNA handling skills and multiple days of effort. To solve this, we developed an ultrashort PRO-seq (uPRO) method that requires minimal steps. In uPRO, the requirement of only two reactions - RNA adaptor ligation and template switch reverse transcription - reduced the procedure into less than a single day. Using uPRO, we generated genome-wide transcription profiles of human haploid cell lines (HAP1) and peripheral blood samples combined with Chromatin Run-On sequencing (pChRO). Blood cell handling procedure is dramatically reduced using pChRO directly on crude chromatin preparations, and enables utilizing archived specimens. As a result, we identified individual differences in the transcriptional profiles of human whole blood from a small volume (~1 ml). We also generated blood cell type specific transcription data, and deconvoluted the nucleated blood cell compositions by modeling to the reference datasets. Overall, uPRO and pChRO provided a powerful platform to identify differentially expressed genes between individuals with minimal sample requirements.