Dissociation of microdissected mouse brain tissue for artifact free single-cell RNA sequencing

Abstract

Summary Single-cell RNA sequencing (scRNA-seq) provides the transcriptome of individual cells and addresses previously intractable problems including the central nervous system’s transcriptional responses during health and disease. However, dissociating brain cells is challenging and induces artificial transcriptional responses. Here, we describe an enzymatic dissociation method for mouse brain that prevents dissociation artifacts and lowers technical variations with standardized steps. We tested this protocol on microdissected brain tissue of 3-week- to 24-month-old mice and obtained high-quality scRNA-seq results. For complete details on the use and execution of this protocol, please refer to Safaiyan et al. (2021) .

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“…Each brain was carefully removed and individually microdissected under a dissection microscope; gray matter was isolated from the frontal cortex and white matter was carefully isolated from the optic tract, medial lemniscus and corpus callosum (attached gray matter and choroid plexus were removed). We used our previously established isolation protocol 63 using gentleMACS with the Neural Tissue Dissociation Kit (Papain; Miltenyi Biotec) and a final concentration of 45 mM actinomycin D (Act-D, Sigma-Aldrich, catalog no. A1410).…”

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“…The whole dissection process was carried out in the span of no more than one hour. The dissected tissues were then frozen at -80°C for either immediate use or later biological assays ( 43 , 44 ).…”

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“…Each brain was removed and under a dissection microscope individually micro-dissected; gray matter was isolated from the frontal cortex and white matter form optic tract, medial lemniscus and corpus callosum (attached gray matter and choroid plexus were carefully removed). We used a microglia isolation protocol we previously described 26 , that prevents ex-vivo transcription and automatizes the mechanical isolation parts using GentleMacs with the Neural Tissue Dissociation Kit (Papain) (Miltenyi Biotec). We added actinomycin D (Act-D, Sigma, No.…”