Lopez-Escobar, L. | bioRxiv Science

bioRxiv2025

Cell motility influences microfluidics capturing in scRNA-seq

Microfluidic isolation methods for single-cell RNA sequencing (scRNA-seq) have primarily been designed for immotile cells, with limited consideration for motile cells, such as those with flagella or cilia. By studying the encapsulation efficiency of the flagellated Trypanosoma brucei using the 10x Genomics platform, we found that maintaining parasites at room temperature results in a low encapsulation yield. We implemented a rapid cooling method to 0{o}C prior to encapsulation, which reduced parasite motility and preventing undesired transcriptomic change. This allowed for a representative scRNA-seq dataset, avoiding the disproportionate loss of the most motile forms. This study highlights the challenges of using motile cells in microfluidic systems and the biases caused by losing specific subpopulations, emphasizing the need for optimized protocols for non-standard mammalian cells. PLAIN LANGUAGE SUMMARYSingle-cell RNA sequencing (scRNA-seq) is a powerful technique used to study individual cells, but most methods have been designed for cells that do not move. This can be a challenge when working with motile cells, like Trypanosoma brucei, a flagellated parasite responsible for sleeping sickness. In our study, we found that these parasites are poorly captured in microfluidic systems, such as the 10x Genomics platform, when kept at room temperature. Their movement reduces the efficiency of encapsulation, leading to biased results. To overcome this, we developed a simple cooling method that quickly lowers the temperature to 0{degrees}C before processing. This reduces parasite motility, improving capture rates while minimizing unwanted changes in gene expression. By applying this approach, we obtained a representative transcriptomic dataset, ensuring that all parasite forms were included. Our findings highlight the importance of adapting microfluidic techniques for motile cells to avoid losing key subpopulations and ensure accurate biological insights.

cell biology↗

bioRxiv2024

m6A landscape is more pervasive when Trypanosoma brucei exits the cell cycle

N6-methyladenosine (m6A) is a mRNA modification with important roles in gene expression. In African trypanosomes, this post-transcriptional modification is detected in hundreds of transcripts and it affects the stability of the variant surface glycoprotein (VSG) transcript in the proliferating blood stream form. However, how m6A landscape varies across the life cycle remains poorly defined. Using full-length, non-fragmented RNA, we immunoprecipitated and sequenced m6A-modified transcripts across three life cycle stages of Trypanosoma brucei - slender (proliferative), stumpy (quiescent), and procyclic forms (proliferative). We found that 1037 transcripts are methylated in at least one of these three life cycle stages. While 21% of methylated transcripts are common in the three stages of the life cycle, globally each stage has a distinct methylome. Interestingly, 47% of methylated transcripts are detected in the quiescent stumpy form only, suggesting a critical role for m6A when parasites exit the cell cycle and prepare for transmission by the Tsetse fly. In this stage, we found that a significant proportion of methylated transcripts encodes for proteins involved in RNA metabolism, which is consistent with their reduced transcription and translation. Moreover, we found that not all major surface proteins are regulated by m6A, as procyclins are not methylated, and that, within the VSG repertoire, not all VSG transcripts are demethylated upon parasite differentiation to procyclic form. This study reveals that the m6A regulatory landscape is specific to each life cycle stage, becoming more pervasive as T. brucei exits the cell cycle. SummaryAfrican trypanosome parasites adapt to mammalian and insect hosts by adjusting gene expression, morphology, and metabolism. In this study, we focus on how N6-methyladenosine (m6A), a post-transcriptional modification, affects the parasites transcriptome throughout its differentiation from the mammalian host to the fly. We found that methylation is differentially regulated as the life cycle progresses, being particularly prevalent in the non-proliferative stumpy form, as more methylated transcripts are found at this insect-infective stage than in slender and procyclic forms. We further show that the not all parasite surface proteins are regulated by m6A and that the previously identified link between m6A methylation and the expression level of the major surface protein of bloodstream forms applies to the active variant surface glycoprotein, but not always to silent genes, suggesting two distinct regulatory mechanisms of (de)methylation.

molecular biology↗