Combining novel scSLAM-seq technology with 10x Genomics Chromium to track microchanges in newly synthesised RNA

Aim

Transcriptional activity at the single-cell level consists of intermittent bursts of transcription that are minutes or hours apart. However, the true temporal dynamics and stochastic nature of subtle transcriptional events remain to be elucidated. Efferocytosis is a process by which dying cells are cleared, and our recent studies show that apoptotic cells are effectively efferocytosed unlike necroptotic cells. Although studies have demonstrated that transcriptional regulation of some specific genes occur during efferocytosis, the precise single-cell transcriptional regulation during efferocytosis is unknown. This proposal aims to combine the novel single-cell technology, scSLAM-seq (single-cell, thio-(SH)-linked alkylation of RNA for metabolic labelling sequencing), with existing 10x Genomics Chromium technologies at IMB to perform cutting-edge analysis of minute transcriptional changes in RNA expression at the single-cell level.

Brief project outline

We propose to develop a novel pipeline for scSLAM-seq analysis combined with 10x Genomics Chromium to metabolically track transcriptional synthesis of new RNA during efferocytosis. We will also develop an improve bioinformatic analysis software by integrating novel GRAND-SLAM and scSplit analysis tools. This streamlined platform will be the first at UQ and Australia to the best of our knowledge, and the technologies developed in this project will be readily available for other researchers through GIH. This versatile technology can be applied to any model organism.

Genomics-based innovative aspect of proposal

The innovation of this proposal essentially lies in developing a novel pipeline based at UQ. This pipeline will be built around the novel methodology, scSLAM-seq, which was recently published in Nature (2019). To the best of our knowledge this technology is yet to be developed at UQ or Australia. Thus, developing this technological pipeline will place UQ as the innovative hub for pioneering cutting-edge genomic technology both nationally and internationally.

Broad applicability of the technique

Here we propose to develop a streamlined pipeline for scSLAM-seq analysis at UQ, where we will establish a start-to-finish full protocol, from performing cell culture assays to scSLAM-seq using cutting edge technology of 10x Genomics Chromium and data analysis. Thus, we anticipate that this technological pipeline, being the first at UQ and Australia (to the best of our knowledge), will place UQ at the forefront in world-class genomic technologies. At the end of this proposal, we envision this technology pipeline to be transferred to IMB Sequencing Facility and GIH, allowing access to all UQ researchers. Users will only need to optimise their individual cell experiments, either in their laboratories or work with GIH technical staff. Once this is optimised, we expect the sequencing and data analysis process to be very rapid and simplified, allowing for the generation of large quantity of genomic data.