RNA-sequencing from single nuclei

Abstract

It has recently been established that synthesis of double-stranded cDNA can be done from a single cell for use in DNA sequencing. Global gene expression can be quantified from the number of reads mapping to each gene, and mutations and mRNA splicing variants determined from the sequence reads. Here we demonstrate that this method of transcriptomic analysis can be done using the extremely low levels of mRNA in a single nucleus, isolated from a mouse neural progenitor cell line and from dissected hippocampal tissue. This method is characterized by excellent coverage and technical reproducibility. On average, more than 16,000 of the 24,057 mouse protein-coding genes were detected from single nuclei, and the amount of gene-expression variation was similar when measured between single nuclei and single cells. Several major advantages of the method exist: first, nuclei, compared with whole cells, have the advantage of being easily isolated from complex tissues and organs, such as those in the CNS. Second, the method can be widely applied to eukaryotic species, including those of different kingdoms. The method also provides insight into regulatory mechanisms specific to the nucleus. Finally, the method enables dissection of regulatory events at the single-cell level; pooling of 10 nuclei or 10 cells obscures some of the variability measured in transcript levels, implying that single nuclei and cells will be extremely useful in revealing the physiological state and interconnectedness of gene regulation in a manner that avoids the masking inherent to conventional transcriptomics using bulk cells or tissues.

Keywords: deep sequencing; nuclear RNA; whole cell RNA.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fluorescence-activated sorting of whole cells and nuclei. (A) NPCs visualized by phase-contrast microscopy (100×). (B) NPCs visualized by epifluorescence microscopy. (C) NPC nuclei visualized by phase-contrast microscopy (100×); demonstrates expected size and morphology. (D) NPC nuclei, examined by epifluorescence microscopy, lack EYFP fluorescence. The white line is a 25-µm calibration ruler. (E) Biparametric flow cytometric analysis of EYFP fluorescence, detected using a 525-nm/25-nm band pass filter, versus side scatter (area signals). The intact cells form a discrete cluster, well separated from cellular debris. The gate (green) designates the region used as the sort window for isolation of single cells. Intact cells lacked red fluorescence, detected using a 620-nm/40-nm band pass filter, when PI was included in the medium. (F) Biparametric flow analysis of PI-stained nuclei using the same instrument settings as in E. The nuclei form a discrete cluster lacking EYFP fluorescence. (G) Biparametric-flow cytometric analysis of PI-stained nuclei, examining red fluorescence, detected using a 620-nm/40-nm band pass filter, versus forward-angle light scatter. The nuclei form a discrete cluster, well separated from cellular debris. (H) Uniparametric display of the 620-nm fluorescence emission from PI-stained nuclei. The window used for sorting single nuclei surrounds the major peak within the nuclear distribution to exclude nuclear aggregates (doublets, triplets, etc.).

Fig. 2.

Quantification of transcript levels in isolated cells and nuclei. Average cycle threshold (Ct) values for eight genes for mouse NPC nuclei (A) and whole cells (B) measured by TaqMan qPCR. Eight replicates of 1, 2, 5, 10, or 100 cells (indicated only for the ActB gene in this figure) were FACS sorted into individual wells in a 384-well plate and used for cDNA synthesis and amplification by PCR. The PCR products were diluted 10-fold and tested for expression of five housekeeping genes (Actb, Eef2, Gapdh, Hsp90ab1, and Rpl13) and three NPC tissue-specific genes (Fabp7, H2afz, and Vim). Ct values of less than 50 were recorded. The number detected for each set of 8 replicate nuclei or cells (error bars) was as follows: (nuclei) Actb—3, 1, 7, 7, and 8 (for 1 nucleus, 2 nuclei, 5 nuclei, 10 nuclei, and 100 nuclei, respectively); Hsp90ab1—4, 2, 7, 8, and 8; Rpl13—7, 8, 8, 8, and 8; Eef2—2, 3, 4, 4, and 8; Vim—4, 6, 7, 8, and 8; Fabp7—5, 4, 8, 8, and 8; H2afz—2, 3, 5, 8, and 8; and Gapdh—6, 5, 7, 8, and 8. (Whole cells) Actb—5, 8, 8, 8, and 8 (for 1 cell, 2 cells, 5 cells, 10 cells, and 100 cells, respectively); Hsp90ab1—6, 8, 8, 8, and 8; Rpl13—6, 8, 8, 8, and 8; Eef2—6, 7, 8, 8, and 8; Vim—7, 6, 8, 8, and 8; Fabp7—6, 8, 8, 8, and 8; H2afz—5, 8, 8, 8, and 8; and Gapdh—5, 8, 8, 8, and 8. The error bars represent the SD of the average Ct measured for each gene locus. The presence of some cells lacking expression for these genes is expected, based on the stochastic activation of gene expression and the current state of knowledge concerning transcriptional bursting within single cells.

Fig. 3.

Isolation of mouse NPCs and nuclei by micromanipulation. (A) Dispersed NPC whole cells. All cells have an intense EYFP signal in the cytoplasm and a DAPI-stained nucleus. (B) Nuclei purified by density-gradient centrifugation. (C) Micromanipulation of a nucleus with a glass capillary. Before micromanipulation, cells and nuclei were visualized by phase-contrast microscopy, and the DAPI and EYFP signals determined by epifluorescence microscopy. (D–G) Whole cells. (H–K) Nuclei. (D and H) Phase contrast. (E and I) DAPI epifluorescence. (F and J) EYFP epifluorescence. (G and K) Stacked phase contrast plus epifluorescence images. (Scale bars: 10 μm in A and B and 25 μm in C, D, and H.)

Fig. 4.

Transcript levels and variation are the same in nuclei and whole cells after sequencing. Expression (RPKM) values for housekeeping genes (A) and NPC-specific genes (B) were used to compare sequenced cells and nuclei. For each gene (x axis), sets of six bars represent the six samples of various numbers of pooled biological triplicates and are in the following order: 1 nucleus, 1 cell, 10 nuclei, 10 cells, 100 nuclei, and 100 cells (indicated for the Fabp7 gene only in B). Error bars denote 1 SD. The y axis is log2 scaled. (C) Measure of dispersion for each group of samples for single and bulk (10 and 100) nuclei and cells. Dispersion (statistical variability) is calculated as the mean of the CV of each gene across biological triplicates.