Apr 11, 2024
Protocol for Nuclei/ Cell Isolation and 10X Genomics Fixed RNA profiling for Human Skeletal Muscle
- 1Buck Institute for research on Aging
Protocol Citation: Nicolas Martin 2024. Protocol for Nuclei/ Cell Isolation and 10X Genomics Fixed RNA profiling for Human Skeletal Muscle. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn68z6l5d/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: April 09, 2024
Last Modified: April 11, 2024
Protocol Integer ID: 97999
Disclaimer
This protocol needs prior approval by the users' institutional review board (IRB) or equivalent ethics committee(s).
Abstract
This is the 10X Genomics protocol to fix, dissociate, and profile RNA from human skeletal muscle tissue.
Guidelines
This protocol needs prior approval by the users' institutional review board (IRB) or equivalent ethics committee(s).
Materials
Refers to the various protocol documents for a complete list of the materials required.
Cell/Nuclei Isolation Protocol for Human Skeletal Muscle
Cell/Nuclei Isolation Protocol for Human Skeletal Muscle
The protocol CG000553 REV B was used to fix, dissociate, and isolate cells/nuclei from frozen human skeletal muscle with the following modifications:
1) 1 mg / mL of Liberase TH was used for dissociation at Step 2b, Page 6.
2) Two extra "spin only" (i.e., steps 3 and 4 ) of the Octodissociator protocol were run for each sample due to the presence of intact/ large tissue pieces at the end of the run at Step 2c, Page 6.
3) Counts were performed using the Cellaca PLX Automated Cell counter (PN: PLX-SYS1) and ViaStain AOPI staining solution (PN: CS2-0106-5mL) at Step 2i on page 6.
Chromium Fixed RNA Profiling Reagent Kits
Chromium Fixed RNA Profiling Reagent Kits
The protocol CG000527 Rev E was used to generate gene expression libraries from fixed cell/nuclei suspension inputs.
Samples were multiplexed in batches of 16 using the user guide CG000565.
Protocol references
The following user guides from 10X Genomics were used for the different steps:
Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling: CG000553 Rev B
Chromium Fixed RNA Profiling Reagent Kits for Multiplexed Samples: CG000527 Rev E
Chromium Fixed RNA Profiling Multiplexed Samples Pooling Workbook: CG000565 RevB