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. 2020 Jan 10;48(1):e4.
doi: 10.1093/nar/gkz968.

FIN-Seq: transcriptional profiling of specific cell types from frozen archived tissue of the human central nervous system

Ryoji Amamoto  1   2 , Emanuela Zuccaro  1 , Nathan C Curry  1 , Sonia Khurana  1 , Hsu-Hsin Chen  1 , Constance L Cepko  2 , Paola Arlotta  1   3
Affiliations

FIN-Seq: transcriptional profiling of specific cell types from frozen archived tissue of the human central nervous system

Ryoji Amamoto et al. Nucleic Acids Res. .

Abstract

Thousands of frozen, archived tissue samples from the human central nervous system (CNS) are currently available in brain banks. As recent developments in RNA sequencing technologies are beginning to elucidate the cellular diversity present within the human CNS, it is becoming clear that an understanding of this diversity would greatly benefit from deeper transcriptional analyses. Single cell and single nucleus RNA profiling provide one avenue to decipher this heterogeneity. An alternative, complementary approach is to profile isolated, pre-defined cell types and use methods that can be applied to many archived human tissue samples that have been stored long-term. Here, we developed FIN-Seq (Frozen Immunolabeled Nuclei Sequencing), a method that accomplishes these goals. FIN-Seq uses immunohistochemical isolation of nuclei of specific cell types from frozen human tissue, followed by bulk RNA-Sequencing. We applied this method to frozen postmortem samples of human cerebral cortex and retina and were able to identify transcripts, including low abundance transcripts, in specific cell types.

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Figures

Figure 1.
Figure 1.
Isolation and transcriptome sequencing of two neuronal subtypes from the frozen mouse neocortex. (A) Schematic of FIN-Seq for frozen adult mouse brain. Nuclei were extracted from the frozen mouse neocortex by Dounce homogenization. The nuclei were fixed and immunolabeled with anti-BCL11B and anti-SATB2 antibodies. Two nuclear populations were isolated by FACS based on expression level of these two proteins. The nuclei were reverse crosslinked by protease digestion, and the RNA was extracted. Sequencing libraries were generated and subsequently sequenced to obtain cell type specific transcriptomes. (B) Representative immunohistochemistry images using BCL11B and SATB2 antibodies in the P30 mouse neocortex showed SATB2 expression in the upper layers and BCL11B expression in the deep layers (Left image). In layer 5, there were sparse cells that express both SATB2 and BCL11B (bottom right image). (C) FACS plot of nuclei labeled with SATB2 and BCL11B antibodies showed a cluster of nuclei immunolabeled with BCL11B and a cluster of nuclei labeled with SATB2. (D) Isolated nuclei were counterstained by the Hoechst dye and either SATB2 or BCL11B in the SATB2+ population (top panels) or BCL11B+ population (bottom panels). (E) Representative quantification of read counts mapped by transcript position (5′ to 3′) for every gene. (F) Representative quantification of percentage of read counts mapped to exonic, intronic, or intergenic regions of the genome. (G) Heatmap of unbiased top 50 differentially expressed genes between SATB2+ and BCL11B+ populations. Known markers of callosal projection neurons (in red) were enriched in the SATB2+ population while known markers of corticofugal projection neurons (in green) were enriched in the BCL11B+ population. Scale bars; 100 μm (B, right panels, D), 500 μm (B, left panel).
Figure 2.
Figure 2.
Isolation and profiling of neuronal subtypes from the frozen human cerebral cortex. (A) Schematic of FIN-Seq for frozen human cerebral cortex. Nuclei were isolated and subsequently fixed in 4% PFA. They were immunolabeled with anti-BCL11B and anti-SATB2 antibodies, and FACS isolated into populations. RNA from the nuclei were sequenced to obtain a cell type specific transcriptome. (B) Representative immunohistochemistry of the adult human cerebral cortex using anti-BCL11B and anti-SATB2 antibodies. Some nuclei expressed both SATB2 and BCL11B (arrows), some nuclei expressed BCL11B but not SATB2 (arrowheads), and many nuclei expressed SATB2 but not BCL11B. (C) A heatmap representing relative expression levels of excitatory neuron markers previously identified by single nuclei RNA sequencing that are differentially expressed (adjusted P-value < 0.05) between SATB2+ and All populations. Markers of neuronal subtype Ex4 (outlined in red), which expresses SATB2, were enriched in the SATB2+ population. (D) Validation of Ex4 markers, COL6A1 (left panel) and ANXA1 (right panel) using RNAscope single molecule FISH. Both COL6A1 and ANXA1 were expressed in SATB2+ neurons (arrows). (E) A heatmap representing relative expression levels of inhibitory neuron markers previously identified by single nuclei RNA sequencing that are differentially expressed (adjusted P-value < 0.05) between BCL11B+ and All populations. Markers of neuronal subtypes, In1, In4, In5 and In6, all of which express BCL11B, were enriched in the BCL11B+ population. Scale bars: 100 μm (B), 50 μm (D).
Figure 3.
Figure 3.
Isolation and sequencing of cone photoreceptor nuclei from the frozen human retina. (A) Schematic of FIN-Seq for the frozen human retina. Nuclei were extracted from the frozen retina and subsequently fixed in 4% PFA. Nuclei were then immunolabeled with an anti-CAR antibody and sorted. CAR+ and CAR− populations were obtained and the nuclear RNA was sequenced. (B) Representative immunohistochemistry of an adult human retina section using the anti-CAR antibody (middle panel) and DAPI (left panel). CAR+ cone photoreceptors were localized to the uppermost layer of the ONL. (C) A heatmap representing relative expression levels of retinal cell type markers previously identified by single nuclei RNA sequencing (adjusted P-value < 1E−10) that are differentially expressed (adjusted P-value < 0.05) between CAR− and CAR+ populations. Cone markers were enriched in the CAR+ population. (D) Validation of new human cone photoreceptor markers by single molecule FISH. Expression levels for DHRS3 and RAB41 from the RNA-seq are indicated in the graphs (left panels). Both DHRS3 and RAB41 were expressed in the ARR3+ cone photoreceptors (arrows). ONL, Outer Nuclear Layer; INL, Inner Nuclear Layer; GCL, Ganglion Cell Layer; MG, Müller Glia; AC, Amacrine Cell; HC, Horizontal Cell; RGC, Retinal Ganglion Cell; BC, Bipolar Cell. Scale bars; 50 μm (B), 10 μm (D).
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References

    1. Cherry T.J., Yang M.G., Harmin D.A., Tao P., Timms A.E., Bauwens M., Allikmets R., Jones E.M., Chen R., DeBaere E. et al. .. Epigenomic profiling and single-nucleus-RNA-seq reveal cis-regulatory elements in human retina, macula and rpe and non-coding genetic variation. 2018; bioRxiv doi:08 September 2018, preprint: not peer reviewed10.1101/412361. - DOI
    1. Darmanis S., Sloan S.A., Zhang Y., Enge M., Caneda C., Shuer L.M., Hayden Gephart M.G., Barres B.A., Quake S.R.. A survey of human brain transcriptome diversity at the single cell level. Proc. Natl. Acad. Sci. U.S.A. 2015; 112:7285–7290. - PMC - PubMed
    1. Hodge R.D., Bakken T.E., Miller J.A., Smith K.A., Barkan E.R., Graybuck L.T., Close J.L., Long B., Penn O., Yao Z. et al. .. Conserved cell types with divergent features between human and mouse cortex. 2018; bioRxiv doi:05 August 2018, preprint: not peer reviewed10.1101/384826. - DOI - PMC - PubMed
    1. Lake B.B., Ai R., Kaeser G.E., Salathia N.S., Yung Y.C., Liu R., Wildberg A., Gao D., Fung H.-L.L., Chen S. et al. .. Neuronal subtypes and diversity revealed by single-nucleus RNA sequencing of the human brain. Science (New York, N.Y.). 2016; 352:1586–1590. - PMC - PubMed
    1. Lake B.B., Chen S., Sos B.C., Fan J., Kaeser G.E., Yung Y.C., Duong T.E., Gao D., Chun J., Kharchenko P.V. et al. .. Integrative single-cell analysis of transcriptional and epigenetic states in the human adult brain. Nat. Biotechnol. 2018; 36:70–80. - PMC - PubMed

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