Oct 07, 2025
Single-nucleus RNA (snRNA) sequencing
- Anita Adami1,2,
- Raquel Garza1,2
- 1Lund University;
- 2Aligning Science Across Parkinson's (ASAP)
Protocol Citation: Anita Adami, Raquel Garza 2025. Single-nucleus RNA (snRNA) sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx41ndl8j/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: October 03, 2025
Last Modified: October 07, 2025
Protocol Integer ID: 228932
Keywords: ASAPCRN, nucleus rna, snrna, workflow for nuclei, rna, nuclei, sequencing step, sequencing workflow
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000520
Aligning Science Across Parkinson's
Grant ID: ASAP-024296
Aligning Science Across Parkinson's
Grant ID: ASAP-025170
Abstract
Step-by-step protocol describing the snRNA sequencing workflow for nuclei isolated from post-mortem human brain tissue.
Troubleshooting
Nuclei isolation from human brain tissue
5h
Isolate nuclei from human brain tissue following the protocol we have previously published:
5h
Single nuclei preparation for sequencing
30m
Load the single-nuclei suspension (8500-10000 nuclei/sample) onto the Chromium Next GEM Chip G Single Cell Kit together with the reverse transcription mastermix following the manufacturer’s instructions. The protocol for the Chromium GEM single cell 3’ kit to generate single-cell gel beads in emulsion can be found on the 10X Genomics website (10X Genomics, PN-1000268). It is possible that new kits and chemistries are released by 10X Genomics. In that case, it can be relevant to use the latest version of the kit and of the protocols provided by the manufacturer.
30m
Amplify cDNA following the guidelines from 10X Genomics. We used 13 cycles of amplification of the 3' libraries.
Generate sequencing libraries with unique dual indices (TT set A).
snRNA sequencing
3d
Pool the libraries from the previous step for sequencing on an Illumina sequencer (we sequenced either on a Novaseq6000 or on a Novaseq X Plus) using a 100-cycle kit and 28-10-10-90 reads.
3d