DiscussionT. brucei, along with other kinetoplastids, has an unusual genome structure with implications for transcription, mRNA processing and gene expression regulation. Genes are transcribed in large tandem arrays from common promoters and are processed by trans splicing of the 5’ cap and polyadenylation to generate mature individual transcripts. This negates gene expression regulation at the level of transcription for nearly all genes. The first part of the talk will focus on R-loops, three stranded nucleic acid structures. Mapping R-loops, via DRIP-seq, genome-wide reveals their association with intergenic sequences between tandemly arranged genes, promoters and repeat sequences. Genetic knockout or knockdown of R-loop processing enzymes RNase H1 and 2A increases levels of R-loops at specific loci. These are associated with specific DNA damage as well as switching of the variant surface glycoprotein, which is essential for immune evasion in the mammalian host. Despite this genome arrangement, T. brucei regulates transcript levels via post-transcriptional mechanisms such as degradation. These transcript levels can be profiled with single cell transcriptomics (scRNA-seq), which will be discussed in the second part of the talk. In the mammal, T. brucei differentiates from replicative slender forms to cell cycle arrested stumpy forms. Using oligopeptides to induce asynchronous differentiation in vitro, scRNA-seq was used to capture individual transcriptomes of slender, stumpy and intermediate stages, allowing a trajectory of differentiation to be reconstructed. Analysis of gene expression dynamics across the trajectory revealed several details of the process. These include: the lack of a discrete intermediate transcriptome; the precise timing of cell cycle exit, immediately prior to late G1; the transient expression of several genes not identified by bulk-analysis; and the expression timing of known and putative differentiation factors during the developmental processes including ZC3H20. The truncated development of ZC3H20 null T. brucei in response oligopeptides was also profiled by scRNA-seq, revealing the position of this regulators action in stumpy development.