A rapid and accurate differential centrifugation method for platelet counts.

Abstract

<h3>ABSTRACT</h3> Spinal motor axons traverse large distances to innervate target muscle, and thus require local control of cellular events for proper functioning of the distal axon. To interrogate axon-specific processes we developed Axon-seq, a refined method incorporating microfluidic devices and stringent bioinformatic quality controls. Axon-seq demonstrates improved sensitivity and accuracy in whole-transcriptome sequencing of axons compared to previously published studies. Importantly, we show that axon transcriptomes are distinct from those of somas, displaying fewer detected genes and no contaminating astrocytic markers. We identified &gt;5,000 transcripts in stem cell-derived spinal motor axons required for local oxidative energy production and ribosome generation. Axons contained unique transcription factor mRNAs, e.g. Ybx1, with implications for local axonal functions. Cross-comparison with peripheral sensory neuron datasets identified a common axon transcriptome consisting of 1,750 mRNAs, as well as 396 mRNAs unique to motor axons, which included genes for ribogenesis, vesicle transport and mRNA splicing. As motor axons degenerate in amyotrophic lateral sclerosis (ALS), we investigated their response to the disease-causing SOD1^G93A mutation, identifying 121 ALS-dysregulated transcripts. Several of these are implicated in axonal and dendritic outgrowth, including Nrp1, Dbn1, and Nek1, a known ALS-causing gene. In conclusion, Axon-seq proves a robust and improved method for RNA-seq of axons, furthers our understanding of peripheral axon biology, and identifies novel therapeutic targets to maintain neural connectivity in disease.