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SUMMARY RNA‐Sequencing is widely used to investigate changes in gene expression at the transcription level in plants. Most plant RNA‐Seq analysis pipelines base the normalization approaches on the assumption that total transcript levels do not vary between samples. However, this assumption has not been demonstrated. In fact, many common experimental treatments and genetic alterations affect transcription efficiency or RNA stability, resulting in unequal transcript abundance. The addition of synthetic RNA controls is a simple correction that controls for variation in total mRNA levels. However, adding spike‐ins appropriately is challenging with complex plant tissue, and carefully considering how they are added is essential to their successful use. We demonstrate that adding external RNA spike‐ins as a normalization control produces differences in RNA‐Seq analysis compared to traditional normalization methods, even between two times of day in untreated plants. We illustrate the use of RNA spike‐ins with 3' RNA‐Seq and present a normalization pipeline that accounts for differences in total transcriptional levels. We evaluate the effect of normalization methods on identifying differentially expressed genes in the context of identifying the effect of the time of day on gene expression and response to chilling stress in sorghum. 

During infections with the malaria parasitesPlasmodium vivax, patients exhibit rhythmic fevers every 48 h. These fever cycles correspond with the time the parasites take to traverse the intraerythrocytic cycle (IEC). In otherPlasmodiumspecies that infect either humans or mice, the IEC is likely guided by a parasite-intrinsic clock [Rijo-Ferreiraet al.,Science368, 746–753 (2020); Smithet al.,Science368, 754–759 (2020)], suggesting that intrinsic clock mechanisms may be a fundamental feature of malaria parasites. Moreover, becausePlasmodiumcycle times are multiples of 24 h, the IECs may be coordinated with the host circadian clock(s). Such coordination could explain the synchronization of the parasite population in the host and enable alignment of IEC and circadian cycle phases. We utilized an ex vivo culture of whole blood from patients infected withP. vivaxto examine the dynamics of the host circadian transcriptome and the parasite IEC transcriptome. Transcriptome dynamics revealed that the phases of the host circadian cycle and the parasite IEC are correlated across multiple patients, showing that the cycles are phase coupled. In mouse model systems, host–parasite cycle coupling appears to provide a selective advantage for the parasite. Thus, understanding how host and parasite cycles are coupled in humans could enable antimalarial therapies that disrupt this coupling. 

The blood stage of the infection of the malaria parasite Plasmodium falciparum exhibits a 48-hour developmental cycle that culminates in the synchronous release of parasites from red blood cells, which triggers 48-hour fever cycles in the host. This cycle could be driven extrinsically by host circadian processes or by a parasite-intrinsic oscillator. To distinguish between these hypotheses, we examine the P. falciparum cycle in an in vitro culture system and show that the parasite has molecular signatures associated with circadian and cell cycle oscillators. Each of the four strains examined has a different period, which indicates strain-intrinsic period control. Finally, we demonstrate that parasites have low cell-to-cell variance in cycle period, on par with a circadian oscillator. We conclude that an intrinsic oscillator maintains Plasmodium ’s rhythmic life cycle.