I am a postdoctoral researcher in the Glioma Genomics group at Leeds Institute of Medical Research (LIMR). The main focus of my work, which is funded by The Brain Tumour Charity, is to understand how glioblastoma (GBM) brain tumours acquire resistance to treatment, leading 100% of primary tumours to recur. The two working hypotheses of our group are that either individual subclones from the primary tumour are inherently resistant and selected for during treatment, or that transcriptional reprogramming enables cells to adapt their phenotype and become resistant.
To understand more about the mechanism of resistance in GBM, I am using a novel technique known as nanobiopsy that allows us to extract cytoplasmic material from single brain tumour cells without killing them. Using this technique we can now, for the first time, longitudinally track and biopsy single cells sequentially during treatment, thereby characterising how the transcriptional profile of individual cells change. To achieve this, I am sequencing sub-single cell RNAseq libraries, created from cytoplasm nanobiopsied pre and post treatment, using the SMART-seq2 and NEXTeraXT workflow.
The biggest challenge that had to be overcome was the amplification of such small amounts of RNA (<1pg) into adequate cDNA for use in NGS library preparation. Dr Iain Macaulay, at the Earlham Institute, assisted with this part of the project. By acquiring a travel grant by the Harold Hyam Wingate Foundation, I was able to visit his state-of-the-art laboratory and worked closely with experts in his single cell technology group to optimise the SMART-seq2 protocol for ultra-low RNA amounts. We have successfully obtained sufficient amounts of cDNA for downstream library preparation, from a starting material of as low as 0.5pg of RNA (approx. 5-10% of a cell’s RNA).
Apart from achieving our very challenging aim, my visit to the Earlham Institute was very inspiring. I spent time preparing RNAseq libraries using different robots, I learnt a quick and easy way to check the sorting alignment on FACS Melody prior to sorting, and I have brainstormed and discussed other alternative ways of amplifying the nanobiopsies in order to get information beyond the coding RNA species.
Earlham Institute: https://www.earlham.ac.uk/
Twitter page: @EarlhamInst