TY  - JOUR
AU  - Ernst, Kati
AU  - Okonechnikov, Konstantin
AU  - Bageritz, Josephine
AU  - Perera, Ashwyn A
AU  - Mallm, Jan-Philipp
AU  - Wittmann, Andrea
AU  - Maaß, Kendra K
AU  - Leible, Svenja
AU  - Boutros, Michael
AU  - Pfister, Stefan M
AU  - Zuckermann, Marc
AU  - Jones, David
TI  - A simplified preparation method for single-nucleus RNA-sequencing using long-term frozen brain tumor tissues.
JO  - Scientific reports
VL  - 15
IS  - 1
SN  - 2045-2322
CY  - [London]
PB  - Springer Nature
M1  - DKFZ-2025-00793
SP  - 12849
PY  - 2025
N1  - #EA:B360#EA:B062#LA:B360#LA:B062#
AB  - Single-cell RNA-sequencing has provided intriguing new insights into research areas such as developmental processes and tumor heterogeneity. Most approaches, however, rely on the availability of fresh surgical specimens, thereby dramatically reducing the ability to profile particularly rare tissue types. Here, we optimized a method to isolate intact nuclei from long-term frozen pediatric glioma tissues. We performed a technical comparison between different single-nucleus RNA-sequencing (snRNA-seq) systems and applied the established nucleus isolation method to analyze frozen primary glioma tissues. The results show that our fast, simple and low-cost nuclear isolation protocol provides intact nuclei, which can be used in both droplet- and plate-based single-cell sequencing platforms - allowing the identification of distinct tumor cell populations and infiltrating microglia. Additional optimization to include shorter RNA fragments in the 3' sequencing library improved gene detection and cell type annotation. Taken together, the method dramatically increases the potential of studying rare tumor entities and is specifically tailored for using frozen brain tumor tissue.
KW  - Humans
KW  - Brain Neoplasms: genetics
KW  - Brain Neoplasms: pathology
KW  - Single-Cell Analysis: methods
KW  - Sequence Analysis, RNA: methods
KW  - Glioma: genetics
KW  - Glioma: pathology
KW  - Cell Nucleus: genetics
KW  - Child
KW  - Freezing
LB  - PUB:(DE-HGF)16
C6  - pmid:40229354
DO  - DOI:10.1038/s41598-025-97053-9
UR  - https://inrepo02.dkfz.de/record/300359
ER  -