Synaptosome Sorting Using Reversible Fixative—A Versatile Approach for Synaptic Transcriptome Sequencing (SynSURF-Seq)

It is generally assumed that presynaptic and postsynaptic transcriptomes closely resemble axonal and dendritic transcriptomes, respectively. By developing a high-precision technique to profile synaptic transcriptomes, we demonstrate this assumption to be incorrect.

Using the cleavable crosslinker dithiobis(succinimidyl propionate) (DSP), which is unreactive against RNAs and circumvents inter-synaptic aggregation compared to conventional paraformaldehyde fixation, we conducted triple sorting via immunofluorescence markers (synaptophysin and PSD-95) and size-gating to minimize glial contaminations. The sorted synaptosome were reductively de-crosslinked to release intact RNAs for next generation sequencing.