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Concept
Patch-sequencing
Résumé
Patch-sequencing (patch-seq) is a method designed for tackling specific problems involved in characterizing neurons. As neural tissues are one of the most transcriptomically diverse populations of cells, classifying neurons into cell types in order to understand the circuits they form is a major challenge for neuroscientists. Combining classical classification methods with single cell RNA-sequencing post-hoc has proved to be difficult and slow. By combining multiple data modalities such as electrophysiology, sequencing and microscopy, Patch-seq allows for neurons to be characterized in multiple ways simultaneously. It currently suffers from low throughput relative to other sequencing methods mainly due to the manual labor involved in achieving a successful patch-clamp recording on a neuron. Investigations are currently underway to automate patch-clamp technology which will improve the throughput of patch-seq as well.
Patch-seq is a specialized form of the patch clamp recording technique, the gold standard of single cell electrophysiological studies for its millisecond resolution of cellular electrophysiology, its ability to detect currents of specific ions, and perhaps most importantly for its ability to form a voltage clamp on the cell membrane. The technique was originally developed using a small glass tube, pipette, which is rapidly heated and stretched to produce a needle like shape with an opening diameter of 3-5 millimeters. Modern preparations use different tip diameters depending on the intended application. The pipette is filled with a salt bath to conduct ionic currents before being pressed onto a cells surface to form an electrical seal with a high electrical resistance (measured in the unit ohm) which ensures low noise in the recording. While this seal will be of the order of megaohms, it has been found that applying slight suction will result in a seal with a resistance greater than a giga-ohm. The high resistance seal allows the experimenter to hold the patch of membrane within the seal at a desired voltage for the study of voltage-gated ion channels.
Source officielle
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