A reliable strategy for single-cell RNA sequencing analysis using cryoconserved primary cortical cells.

Animals Base Sequence Cryopreservation Flow Cytometry Mice Sequence Analysis, RNA Single-Cell Analysis
Cryopreservation E15.5 Embryonic mouse brain Primary cortical cells Single-cell RNA sequencing

Journal

Journal of neuroscience methods
ISSN: 1872-678X
Titre abrégé: J Neurosci Methods
Pays: Netherlands
ID NLM: 7905558

Informations de publication

Date de publication:
01 01 2021
Historique:
received: 29 04 2020
revised: 30 07 2020
accepted: 23 09 2020
pubmed: 29 9 2020
medline: 22 6 2021
entrez: 28 9 2020
Statut: ppublish

Résumé

The application of single-cell RNA sequencing (scRNASeq) represents a unique approach to identify hundreds to millions of cells in mammalian cortical multilayers at different stages of embryogenesis. ScRNASeq technology applied to neurological studies requires the use of fresh starting materials because standard cryopreservation methods do not guarantee high viability of cortical primary cells derived from dissected brain areas. Here we set up and validate an innovative strategy to perform scRNASeq studies in cryopreserved primary cortical cells isolated from E15.5 mouse embryo. In order to freeze cortical primary cells, we have employed Neurostore, a medium able to guarantee high viability and cell composition of embryonic cortex after thawing. We showed for the first time the possibility to run scRNASeq experiments on primary cortical cells in an off-line set-up, ensuring cellular integrity and diversity. By trypan blue assay and flow cytometry analysis, we found that Neurostore-cryopreserved cortical cells showed approximately 95 % of viability. Satisfactory RNA recovery and cDNA libraries were achieved. Transcriptome sequencing of 35,763 cryoconserved single cells yielded a robust data-set, identifying 25 cell clusters in three biological samples. Prevalence of peculiar neural populations before and after the cryopreservation-resuscitation procedure was verified by marker gene expression and immunofluorescence analysis. Our findings support the evidence that frozen primary cortical cells can be successfully employed in scRNASeq experiments allowing an unprecedented flexibility in experimental procedures, such as sample preparation and subsequent processing steps performed in different locations.

Sections du résumé

BACKGROUND
The application of single-cell RNA sequencing (scRNASeq) represents a unique approach to identify hundreds to millions of cells in mammalian cortical multilayers at different stages of embryogenesis. ScRNASeq technology applied to neurological studies requires the use of fresh starting materials because standard cryopreservation methods do not guarantee high viability of cortical primary cells derived from dissected brain areas.
NEW METHOD
Here we set up and validate an innovative strategy to perform scRNASeq studies in cryopreserved primary cortical cells isolated from E15.5 mouse embryo. In order to freeze cortical primary cells, we have employed Neurostore, a medium able to guarantee high viability and cell composition of embryonic cortex after thawing.
COMPARISON WITH EXISTING METHODS
We showed for the first time the possibility to run scRNASeq experiments on primary cortical cells in an off-line set-up, ensuring cellular integrity and diversity.
RESULTS
By trypan blue assay and flow cytometry analysis, we found that Neurostore-cryopreserved cortical cells showed approximately 95 % of viability. Satisfactory RNA recovery and cDNA libraries were achieved. Transcriptome sequencing of 35,763 cryoconserved single cells yielded a robust data-set, identifying 25 cell clusters in three biological samples. Prevalence of peculiar neural populations before and after the cryopreservation-resuscitation procedure was verified by marker gene expression and immunofluorescence analysis.
CONCLUSIONS
Our findings support the evidence that frozen primary cortical cells can be successfully employed in scRNASeq experiments allowing an unprecedented flexibility in experimental procedures, such as sample preparation and subsequent processing steps performed in different locations.

Identifiants

DOI: 10.1016/j.jneumeth.2020.108960 PMID: 32987100
pubmed: 32987100
pii: S0165-0270(20)30383-6
doi: 10.1016/j.jneumeth.2020.108960
pii:
doi:

Types de publication

Journal Article Research Support, Non-U.S. Gov't

Langues

eng

Sous-ensembles de citation

IM

Pagination

108960

Informations de copyright

Copyright © 2020 Consiglio nazionale delle ricerche CNR Italy. Published by Elsevier B.V. All rights reserved.

Auteurs

Lucia Verrillo (L)

Institute of Genetics and Biophysics "Adriano Buzzati-Traverso", National Research Council, Naples, Italy; University of Campania "Luigi Vanvitelli", Caserta, Italy.

Eleonora Mangano (E)

Institute of Biomedical Technologies, National Research Council, Segrate, Milan, Italy.

Denise Drongitis (D)

Institute of Genetics and Biophysics "Adriano Buzzati-Traverso", National Research Council, Naples, Italy. Electronic address: denise.drongitis@igb.cnr.it.

Ivan Merelli (I)

Institute of Biomedical Technologies, National Research Council, Segrate, Milan, Italy.

Francesca Pischedda (F)

University of Trento, CIBIO & Dulbecco Telethon Institute, Trento, Italy.

Giovanni Piccoli (G)

University of Trento, CIBIO & Dulbecco Telethon Institute, Trento, Italy.

Clarissa Consolandi (C)

Institute of Biomedical Technologies, National Research Council, Segrate, Milan, Italy.

Roberta Bordoni (R)

Institute of Biomedical Technologies, National Research Council, Segrate, Milan, Italy.

Maria Giuseppina Miano (MG)

Institute of Genetics and Biophysics "Adriano Buzzati-Traverso", National Research Council, Naples, Italy. Electronic address: mariag.miano@igb.cnr.it.

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Classifications MeSH

questionsmedicales.fr › Eucaryotes › Animaux › A reliable strategy for single-cell RNA...
questionsmedicales.fr › Sciences de l'information › Sources d'information › Services d'information › Documentation › Données de séquences moléculaires › Séquence nucléotidique › A reliable strategy for single-cell RNA...
questionsmedicales.fr › Techniques d'investigation › Conservation biologique › Cryoconservation › A reliable strategy for single-cell RNA...
questionsmedicales.fr › Techniques d'investigation › Techniques cytologiques › Cytophotométrie › Cytométrie en flux › A reliable strategy for single-cell RNA...
questionsmedicales.fr › Eucaryotes › Animaux › Chordés › Vertébrés › Mammifères › Eutheria › Rodentia › Muridae › Murinae › Souris › A reliable strategy for single-cell RNA...
questionsmedicales.fr › Techniques d'investigation › Techniques génétiques › Analyse de séquence › Analyse de séquence d'ARN › A reliable strategy for single-cell RNA...
questionsmedicales.fr › Techniques d'investigation › Techniques cytologiques › Analyse sur cellule unique › A reliable strategy for single-cell RNA...