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

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

doi:10.1093/nar/gkz968

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.

Highlights

The human central nervous system (CNS) comprises an extremely diverse set of cell types. While this heterogeneous cellular composition has been appreciated since the work of early anatomists, it was not until recently, with the advent of single cell and single nucleus RNA sequencing, that different cell types of the adult human cerebral cortex and retina have begun to be defined at the molecular level (Cherry et al, 2018; Darmanis et al, 2015; Hodge et al, 2018; Lake et al, 2016; Lake et al, 2018; Liang et al, 2019; Lukowski et al, 2018; Peng et al, 2019; Phillips et al, 2018)
We aimed to develop a method that enables bulk RNA sequencing of specific cell types and extends to archived frozen tissue
We found that all Callosal Projection Neurons (CPN) markers were enriched in the SATB2+ population and all Corticofugal Projection Neurons (CFuPN) markers were enriched in the BCL11B+ population (Figure 1-figure supplement 2)

Summary

INTRODUCTION

The human central nervous system (CNS) comprises an extremely diverse set of cell types. Nuclei Sequencing), a technology that combines nuclear isolation, fixation, immunolabeling, FACS, and RNA sequencing to obtain the gene expression profile of specific neuronal subtypes from frozen, archived human CNS tissue While some antibodies such as those against NeuN and SOX6 are known to work with fresh tissue (Kozlenkov et al, 2018), a method to apply a wider range of antibodies against cell type specific markers is not available. We found that the nuclear transcripts captured with FIN-Seq represented more of the whole-cell transcripts compared to single nucleus sequencing This is a novel, cost-effective technology that could enable deep transcriptional analysis of user-defined cell types from widelyavailable frozen human CNS samples

RESULTS

DISCUSSION

Findings

MATERIALS AND METHODS