Project description:Previously, transcriptomics data for mollusc has been obtained by whole-brain bulk RNA-seq and low-throughput scRNA-seq. We want to construct the first molluscan high-throughput single-neuron transcriptomes for Berghia stephanieae. Around 129,000 cells were collected from 20 brains and the libraries were constructed using the 10X Genomics' Chromium platform. The brains were separated into two samples: the main brain (i.e., the cerebropleural, pedal, buccal ganglion) and the rhinophore ganglion sample. After library preparation, around 1,000 cells were receovered and sequenced. After data analysis, the cells formed eight clusters with marker genes for each cluster identified. Various cell populations that express a wide range of both small-molecule neurotransmitters and neuropeptides such as serotonergic, small cardioactive peptide (SCP), APGWamide, and FMRFamide cells were also identified in the dataset. Interestingly, cells from the rhinophore ganglion of Berghia exhibit great cell heterogeneity, with cells splitting into two general categories and four distinct clusters. The project produced a single-cell dissociation protocol that can be adapted for use in other nudibranch molluscs and a custom data analysis pipeline for data of this nature.
Project description:Recombinant human erythropoietin administration studies involving transcriptomic approaches have demonstrated a gene-expression signature that could aid detection of blood doping. However, current anti-doping testing does not involve blood collection into tubes with RNA preservative. This study investigated if whole blood in long-term storage and whole blood leftover from standard haematological testing in short-term storage could be used for transcriptomic analysis despite lacking RNA preservative. Whole blood samples were collected from thirteen and fourteen healthy males, for long-term and short-term storage experiments. Long-term storage: whole blood collected into Tempus™ tubes and K2EDTA tubes and subjected to long-term (i.e., −80°C) storage and RNA extracted. After storage, K2EDTA tubes were thawed and extracted using GeneJET RNA Purification Kit (Thermo Fisher Scientific, Vilnius, Lithuania) or Tempus™ Spin RNA Isolation Kit (Life Technologies, Carlsbad, CA, USA). RNA quality and purity was sufficient for gene expression analysis. Principle Component Analysis of microarray and RNA-seq gene expression data for long-term storage: When comparing gene expression between blood tubes with and without RNA preservation, 6% (4058 transcripts) were differentially expressed. RNA quantity, purity and integrity was not significantly compromised from long-term storage in blood storage tubes lacking RNA preservative, indicating that transcriptomic analysis could be conducted using anti-doping samples collected or biobanked without RNA preservation.