# Protocol to dissect and dissociate the mouse brainstem for single-cell RNA-seq applications

## TL;DR

This paper provides a detailed protocol for dissecting and processing the mouse brainstem to prepare cells for single-cell RNA sequencing, especially for small brain regions.

## Contribution

The paper introduces a novel, adaptable protocol for dissociating and preserving mouse brainstem cells for transcriptomic analysis.

## Key findings

- The protocol enables asynchronous sample collection and cryopreservation of brainstem cells from neonatal mice.
- It is optimized for SmartSeq3 library preparation and can be adapted for other brainstem regions and methods.

## Abstract

Processing dissociated cells for transcriptomics is challenging when targeting small brain structures, like brainstem nuclei, where cell yield may be low. Here, we present a protocol for dissecting, dissociating, and cryopreserving mouse brainstem that allows asynchronous sample collection and downstream processing of cells obtained from brainstem tissue in neonatal mice. Although we demonstrate this protocol with the isolated preBötzinger complex and downstream SmartSeq3 cDNA library preparation, it could be readily adapted for other brainstem areas and library preparation approaches.

•Steps for taking transverse slices of the medulla oblongata that contain the preBötC•Detailed punchout procedure for preBötC single-cell dissociation•Asynchronous collection, fixation, and methanol cryopreservation•Optimized for SmartSeq3 cDNA library preparation and FACS

Steps for taking transverse slices of the medulla oblongata that contain the preBötC

Detailed punchout procedure for preBötC single-cell dissociation

Asynchronous collection, fixation, and methanol cryopreservation

Optimized for SmartSeq3 cDNA library preparation and FACS

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Processing dissociated cells for transcriptomics is challenging when targeting small brain structures, like brainstem nuclei, where cell yield may be low. Here, we present a protocol for dissecting, dissociating, and cryopreserving mouse brainstem that allows asynchronous sample collection and downstream processing of cells obtained from brainstem tissue in neonatal mice. Although we demonstrate this protocol with the isolated preBötzinger complex and downstream SmartSeq3 cDNA library preparation, it could be readily adapted for other brainstem areas and library preparation approaches.