A protocol for sequencing the transcriptome of a single nuclei

A protocol is described for sequencing the transcriptome of a cell nucleus. Nuclei are isolated from specimens and sorted by FACS, cDNA libraries are constructed and RNA-seq is performed, followed by data analysis. Some steps follow published methods (Smart-seq2 for cDNA synthesis and Nextera XT barcoded library preparation) and are not described in detail here.

Previous single-cell approaches for RNA-seq from tissues include cell dissociation using protease treatment at 30 °C, which is known to alter the transcriptome. In this protocol, nuclei are isolated at 4 °C from tissue homogenates, which cause minimal damage. Nuclear transcriptomes can be obtained from postmortem human brain tissue stored at -80 °C, making brain archives accessible for RNA-seq from individual neurons. The method also allows investigation of biological features unique to nuclei, such as enrichment of certain transcripts and precursors of some noncoding RNAs. By following this procedure, it takes about 4 d to construct cDNA libraries that are ready for sequencing.

Single nuclei isolation experimental workflow

rna-seq

Dounce homogenization in lysis buffer is used to disrupt cellular membranes for fresh or frozen tissue (a). Nuclei quality and yield is determined by hemocytometer count (b). (ce) Nuclei and cellular debris are filtered for optional purification and immunostaining steps (density gradient centrifugation (c) or staining for neuronal enrichment (d)), or for FACS sorting (e). (f,g) Subsequently, lysis of the nuclei and cDNA synthesis is carried out using either published methods or commercial kits (SMARTer, Clontech) (f), and it is quality-controlled for size distribution using a Bioanalyzer (Agilent) and the presence of several transcripts by qPCR (g). (h) Sequencing and data analysis confirm single nucleus transcriptome capture. Step numbers indicate the corresponding step numbers in the PROCEDURE section. Graphs in g and h are for illustrative purposes only.

Krishnaswami SR, Grindberg RV, Novotny M, Venepally P, Lacar B, Bhutani K, Linker SB, Pham S, Erwin JA, Miller JA, Hodge R, McCarthy JK, Kelder M, McCorrison J, Aevermann BD, Fuertes FD, Scheuermann RH, Lee J, Lein ES, Schork N, McConnell MJ, Gage FH, Lasken RS. (2016) Using single nuclei for RNA-seq to capture the transcriptome of postmortem neurons. Nat Protoc 11(3):499-524. [abstract]

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