Frozen tissue (~25 mg) from either frontal cortex or cerebellar hemisphere (see table of demographics) was dissected at −20 and subjected to dounce homogenization followed by sucrose gradient centrifugation as previously described. Tissue was primarily BA10 or lateral cerebellar hemisphere although some samples were named only as frontal cortex or cerebellum. Nuclei were filtered and incubated for 5 min in 1:1,000 Hoechst (Invitrogen H3569, Waltham MA). A total of 10,000 Hoechst + nuclei from the suspension were then sorted directly into 10× Genomics RT buffer (Pleasanton, CA) on a chilled plate holder to remove doublets, debris, and dying nuclei on a FACS Aria (BD Biosciences, Franklin Lakes, NJ) with a low-pressure nozzle (SI Appendix, Fig. S2 ). Following sorting, reverse transcriptase enzyme was added on ice, and nuclei were immediately processed for encapsulation in the 10× Chromium controller. cDNA and libraries were prepared according to the 10× documentation protocol for 3′ gene expression v3 chemistry.
Genomics rt buffer
Manufactured by BD
Genomics RT buffer is a reagent used in reverse transcription (RT) reactions during genomic analysis. It provides the necessary components to facilitate the conversion of RNA into complementary DNA (cDNA) molecules, which can then be utilized for further downstream applications such as PCR or sequencing.
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2 protocols using genomics rt buffer
1
Single-nucleus RNA-seq from post-mortem brain
2
Single-nucleus RNA-seq from frozen brain
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Frozen tissue (~ 25 mg ) from either section 1 frontal cortex or section 5 cerebellar hemisphere (see table of demographics) was dissected at -20 and subjected to dounce homogenization followed by sucrose gradient centrifugation as previously described. Nuclei were filtered and incubated for 5 minutes in 1:1000 Hoechst (Invitrogen H3569, Waltham MA). 10,000 Hoechst + nuclei from the suspension were then sorted directly into 10x Genomics RT buffer (Pleasanton, CA) on a chilled plate holder to remove doublets, debris, and dying nuclei on a FACS Aria (BD Biosciences, Franklin Lakes, NJ) with low pressure nozzle (Appendix Figure 2). Following sorting, reverse transcriptase enzyme was added on ice, and nuclei were immediately processed for encapsulation in the 10x Chromium controller. cDNA and libraries were prepared according to the 10x documentation protocol for 3' gene expression. Libraries were prepared in matched batches and represent over 16 individual days of sample preparation and 4 separate sequencing runs.
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