Background
Crucian carp (Carassius carassius) is a fish that, unlike most other vertebrates, can survive for a long time without oxygen (anoxia). This ability is necessary for the crucian carp to survive in Northern Europe in small ponds, where the oxygen disappears when ice and snow prevent oxygen to enter from the air or photosynthesis. The fish survives anoxia with the help of a number of physiological adaptations. For example, lactic acid from glycolysis is converted to ethanol, which is washed out of the blood over the gills.
In other vertebrates, the brain in particular is very sensitive to a lack of oxygen. Although we know quite a bit about how the crucian carp can physiologically survive without oxygen, there are still many questions that remain, especially for the brain. We do know, however, that there is a large response at the transcriptome level – thousands and thousands of genes have altered mRNA abundance in response to anoxia (Fig. 1). But without spatial and cellular context, it is challenging to pinpoint the role of all these alterations, and the question arise:
Master project
Aims
The primary aim of the masters project will be to 1a) identify and annotate the clusters corresponding to different cells types, 1b) annotate the clusters with putative cell-type identity, and 1c) investigate how the individual cell types responds to anoxia. A secondary aim, if time permits, will be to use the single-nuclei data for cell-type deconvolution of 2a) an existing spatial transcriptome, and 2b) existing transcriptomes from bulk sequencing of the brain.
Methodology
We have used a state-of-the are modern sequencing technique, single-cell nuclei sequencing, on brain tissue samples from 6 normoxic and 6 anoxic crucian carp. Specifically, we used the split-pool combinatorial barcoding approach with the Parse Evercode WT kit, targeting 100 000 nuclei. This information can be used to identify different cell types and how they alter gene expression during and after anoxia exposure.
The single-nuclei mRNA libraries have already been prepared and sequenced, so the starting point of the project is the raw sequencing data (.fastq.gz files). The student will be tasked with the analysis of the dataset, from mapping and gene-cell matrix generation using the Parse pipeline and/or the Parse software Trailmaker. The data matrix will then be further analysed using for example the Seurat package in R.
Student interests and prerequisites
As the project primarily involves bioinformatical data analysis, it is suited for a student with a keen interest in bioinformatics and working with big datasets. It is also necessary with basic coding skills, as the initial work on the high-performance computing cluster Saga will be done using the Unix language (bash) and remaining analyses and plotting will be done using different packages in R.
Supervisors and research group
Main supervisor will be Sjannie Lefevre (FYSCELL) with co-supervision by Naomi Croft Gusland. Using a wide range of sequencing techniques, Sjannie is investigating the molecular mechanisms of tolerance to anoxia and in particular re-oxygenation in crucian carp, and also the evolution of anoxia tolerance. Naomi is a member of the comparative immunology group and has used single-cell sequencing to investigate the peculiar immune system of the Atlantic cod. Other members of the Lefevre-Nilsson research group are Laura Marian Valencia-Pesqueira, a PhD student working with the crucian carp genome and Lucie Gerber, a research working on crucian carp mitochondrial function. We also have G?ran Nilsson, a professor with a long career in crucian carp research and a current interest in mitochondrial and metabolic adjustments. Our research group offers a welcoming environment, where everyone shows an interest in helping and supporting other members, for example during regular lab meetings (every second week), afternoon tea breaks, and lunching together in the FYSCELL lunchroom, or grabbing a pizza at Frederikke.
Are you interested? Contact Sjannie (s.l.nilsson@ibv.uio.no) and ask for a meeting.