Oct 01, 2025
Multiome sequencing murine ovaries
- David Arboleda-Prado1,
- Benjamin Cosgrove1
- 1Cornell University
Protocol Citation: David Arboleda-Prado, Benjamin Cosgrove 2025. Multiome sequencing murine ovaries. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg317z1l25/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: September 30, 2025
Last Modified: October 01, 2025
Protocol Integer ID: 228592
Keywords: mouse ovary, sequencing murine, protocol for single nuclei, single nuclei
Abstract
Protocol for single nuclei multiome sequencing - mouse ovaries.
Troubleshooting
Ovary collection and nuclei isolation
Female mice were staged for estrous cycles, and both ovaries were collected at diestrus.
Ovaries were immediately flash frozen in liquid nitrogen and stored at −80 °C until use.
For single-nuclei isolation, frozen ovaries were processed using the Chromium Nuclei Isolation Kit (10x Genomics, Pleasanton, CA) following the manufacturer’s protocol for frozen tissue.
Nuclei were washed, counted using an automatic hemocytometer, and resuspended in Diluted Nuclei Buffer (10x Genomics) to the concentration recommended for Multiome library preparation.
Single-nuclei Multiome library preparation and sequencing
Nuclei were loaded on the Chromium Next GEM Chip J using the Chromium Single Cell Multiome ATAC + Gene Expression kit (10x Genomics, Pleasanton, CA), following the manufacturer’s instructions.
The targeted recovery was 10000 nuclei per sample.
Libraries were sequenced on a NovaSeq X platform (Illumina, San Diego, CA). Gene expression (GEX) and ATAC libraries were run across NovaSeqX-25B lanes.