Jun 22, 2026
RNAlater-compatible protocol for nuclei isolation from radical prostatectomy prostate cancer resections, enabling single-nucleus resolution RNA-seq
- Magdalena Krystkiewicz-Orzechowska1,
- Dorota Anusewicz1,
- Katarzyna Kośla1,
- Izabela Baryła1,
- Agata Sakowicz2,
- Michał Olczak3,
- Mateusz Mroczek3,
- Marek Lipiński3,
- Andrzej K. Bednarek1
- 1Department of Molecular Carcinogenesis, Medical University of Łódź;
- 2Department of Molecular Biotechnology, Medical University of Łódź;
- 3II Clinic of Urology, Medical University of Łódź
Protocol Citation: Magdalena Krystkiewicz-Orzechowska, Dorota Anusewicz, Katarzyna Kośla, Izabela Baryła, Agata Sakowicz, Michał Olczak, Mateusz Mroczek, Marek Lipiński, Andrzej K. Bednarek 2026. RNAlater-compatible protocol for nuclei isolation from radical prostatectomy prostate cancer resections, enabling single-nucleus resolution RNA-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g77r93gwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol, and it's working.
Created: December 28, 2025
Last Modified: June 22, 2026
Protocol Integer ID: 235955
Keywords: nucleus resolution rna, preserved prostate cancer, nucleus rna, radical prostatectomy prostate cancer resection, compatible protocol for nuclei isolation, grade recovery of diverse cell state, cell genomics due to the technical challenge, transcriptomic profiling, cell genomic, prostate cancer, rnalater, diverse cell state, concordant upregulation of nucleus, genuine metabolic reprogramming, nuclei isolation, effective strategy for translational oncology, quality nuclei
Funders Acknowledgements:
Medical University of Łódź
Grant ID: 503/0-078-02/503-01-001
Disclaimer
Full study has been published in Scientific Reports and is available: https://doi.org/10.1038/s41598-026-57993-2
Please, cite the article if referring to the protocol:
Krystkiewicz-Orzechowska, M.J., Anusewicz, D., Kośla, K. et al. RNAlater-compatible protocol for nuclei isolation from radical prostatectomy prostate cancer resections, enabling single-nucleus resolution RNA-seq. Sci Rep (2026). https://doi.org/10.1038/s41598-026-57993-2
The datasets generated and analysed in the current study are available via the Broad Institute Single Cell Portal (accession ID: SCP3622).
Abstract
The vast archives of RNAlater-preserved clinical tissues remain largely inaccessible for single-cell genomics due to the technical challenges of isolating high-quality nuclei from fixed material. Current methods often rely on fresh tissue or complex sorting procedures, which limit the applicability of retrospective studies. Here, we present a streamlined, sorter-free single-nucleus RNA sequencing (snRNA-seq) protocol validated on RNAlater-preserved prostate cancer (PC) resections. By coupling bead-mill homogenization with high-salt lysis, we successfully isolated intact nuclei, achieving high library quality (Q30 ≥ 90%) and low ambient noise without the need for fluorescence-activated cell sorting (FACS) or density gradients. Transcriptomic profiling of 3,365 nuclei from low- and high-grade PC specimens resolved eight distinct cellular compartments, faithfully retaining fragile luminal, stromal, and immune lineages. Notably, we demonstrate that elevated mitochondrial read counts in high-grade tumors reflect genuine metabolic reprogramming, evidenced by the concordant upregulation of nucleus-encoded OXPHOS genes, rather than technical artifacts. This atlas-grade recovery of diverse cell states confirms the method’s fidelity and biological resolution. Our approach unlocks the potential of biobanked specimens for routine single-cell analysis, offering a scalable, cost-effective strategy for translational oncology.
Materials
REAGENTS
Homogenization procedure
1x PBS pH 7.4
UFO beads ø 3.5 mm (SSUFO35, Next Advance, Inc.)
Lysis buffer
10 mM Tris-HCl pH 7.4
250 mM NaCl
21 mM MgCl₂
1 mM CaCl₂
1% NP-40
1 mM EGTA
5 mM EDTA
Nuclease-free water
0.2 U/µL RNAse inhibitor cocktail (Millipore-Sigma no. 3335399001)
Wash and resuspension buffers
1x PBS pH 7.4
2% Bovine Serum Albumin solution
0.2 U/µL RNAse inhibitor cocktail (Millipore-Sigma no. 3335399001)
Staining (Pappenheim’s method)
May-Grünwald solution
Giemsa stock solution
EQUIPMENT
Bullet Blender® 5E Pro (Next Advance, Inc.)
Cell stainer ø 40 µm pore PP/Nylon sterile
Bench top centrifuge
Countess™ II FL automated cell counter
Countess™ Cell counting chamber slides
Trypan blue
Collection of samples, and processing of prostate cancer tissues
Examine the post-surgical material microscopically and lesions identified prior to the operation as a tumor (prostate MRI and TRUS biopsy).
Sample lesions using scissors in a way that spares the local structure of a prostate gland.
After resection, immediately transfer a specimen of approximately 5 cm³ to a 1.5 mL RNAlater buffer and store at 4°C until further processing.
Nuclei isolation procedure
Mince a sample into approximately 0.5 mm pieces with a sterile scalpel blade and transfer to 5 mL tube with 2 mL of PBS and 4 UFO beads.
Homogenize with conditions set to speed 20 for 4 minutes in 4°C.
Remove the bullets and centrifuge the disintegrated sample (1500 rpm, 10 min, 4°C).
Remove supernatant.
Perform the lysis in 5 mL of lysis buffer for 50 minutes on ice.
Centrifuge the sample on a sterile cell strainer (ø40 µm) in a lysis buffer (500 rpm, 10 min, 4°C).
The resulting pellet of nuclei suspend in 500 µL of washing buffer with RNase inhibitors cocktail (0.2 U/µL) and 10% DMSO for further freezing and long-term storage in -80°C for downstream purposes.
Collect additional amount of 25 µL intended for morphological assessment of nuclei quality (Pappenheim method) and counting with trypan blue (Countess™ II FL Automated Cell Counter for determining nuclei concentration).
Assessment of the nuclei morphology and quality of sample
Approx. 10 µL of nuclei suspension was smeared on the glass slide for light microscopy.
Dry the smear.
Stain the smear with May Grünwald reagent for 7 minutes.
Gently remove the stain and wash 10 seconds with distilled water.
Stain the specimen with Giemsa reagent (1:10) for 5 minutes.
Gently wash with distilled water and leave until completely dry.
Examine microscopic slides with stained nuclei content under a light microscope using an oil immersion objective at a total magnification of 1000x.
Isolated nuclei stained with Pappenheim method (oil immersion objective at 100x magnification and an ocular at 10x magnification, a total of 1000x magnification; scale bar 10 µm).
Protocol references
10x Genomics protocol for nuclei isolation (10x Genomics demonstrated protocol CG000124 Rev F)