Aug 30, 2024
JAX-Sen: Mouse heart dissociation for single-cell RNA sequencing
- Ramalakshmi Ramasamy1,
- Juliana Alcoforado Diniz1,
- Patrick Fleming1,
- Jessica Garofalo1,
- Paul Robson1,2
- 1The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA;
- 2Department of Genetics and Genome Sciences, University of Connecticut School of Medicine, Farmington, CT, USA
- Cellular Senescence Network (SenNet) Method Development Community
Protocol Citation: Ramalakshmi Ramasamy, Juliana Alcoforado Diniz, Patrick Fleming, Jessica Garofalo, Paul Robson 2024. JAX-Sen: Mouse heart dissociation for single-cell RNA sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzj55xlx1/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: July 02, 2024
Last Modified: August 30, 2024
Protocol Integer ID: 102774
Keywords: JAX-Sen
Funders Acknowledgement:
National Institute on Aging (NIA) JAX-Sen Senescence Tissue Mapping Center Grant
Grant ID: ID: U54 AG079753
Abstract
These samples are part of the JAX-Sen project in the SenNet Consortium. We aim to study and characterize senescence in the C57Bl/6 mouse heart. We compare young (6 months old) and old (24 months old) mouse hearts using scRNAsequencing. This protocol describes the single-cell dissociation of the hearts before library preparation and sequencing.
Reagents and Materials
Reagents and Materials
- 5 ml Protein lobind tubes
- 1.5 or 2 ml Protein lobind tubes
- Micro scissors/ scalpel
- Ice-cold PBS
- Wide-bore pipette tips
- 0.25% Trypsin/EDTA (Gibco, 25200056). - 2 ml/sample
- 20 mg/mL collagenase A and B (Sigma, 10103578001, 11088807001) – 2 ml/sample
- DMEM + 10% FBS
- 100 µm cell strainer (Corning, 431752)
- HBSS (Ca2+/Mg2+ free) (Gibco, 14170120) – 2 ml/sample
- ACK lysis buffer – 2 ml/sample
- SS_buffer – 5 ml/sample
- 40 um cell strainer
- 70 uM strainer
SS_buffer
| A | B | C | D | E | F | G | H | I | |
| Stock conc. | Working conc. | 50 ml | 100 ml | 250 ml | 500 ml | 20 ml | |||
| EDTA | 0.5 M | 2 mM | 0.2 | 0.4 | 1 | 2 | 0.08 | ml | |
| BSA | 10% | 2% | 10 | 20 | 50 | 100 | 4 | ml | |
| FBS | 100% | 15% | 7.5 | 15 | 37.5 | 75 | 3 | ml | |
| DPBS | 1X | make up | 32.2 | 64.4 | 161 | 322 | 12.88 | ml |
Procedure
Procedure
Collect shipment from the warehouse.
Fill up ice buckets and prep them with reagents:
Bucket 1(small): PBS, HBSS, DMEM+FBS, SS_buffer
Bucket 2 (large): For mincing tissue
Confirm if the sample is cold. Take pictures.
Place tubes on ice and mince the tissue (<1mm) with sterile micro scissors.
After mincing, transfer the sample into labeled 5 ml tubes.
Centrifuge at 500 x g, 4°C, 5 min to get rid of the supernatant.
Resuspend the pellet in ice-cold PBS (Ca+ and Mg+ free) and pipette up and down gently with wide-bore pipette tip.
Centrifuge at 500 x g, 4°C, 5 min to get rid of the supernatant.
Resuspend the pellet in ice-cold PBS (Ca+ and Mg+ free) and pipette up and down gently with wide-bore pipette tip.
Centrifuge at 500 x g, 4°C, 5 min to get rid of the supernatant.
Add 2 ml of 0.25% Trypsin/EDTA to the pellet and enzymatically digest at 37°C for 10 min with slow rocking. Check at 5 mins for dissociation, under the microscope.
Stop the digestion by adding an equal volume (1ml) of DMEM/10% FBS.
Centrifuge at 500 x g, 4°C, 5 min to get rid of the supernatant.
Resuspend the pellet 2 ml of 20 mg/mL collagenase A and B mixture and incubate the samples at 37 °C until the tissues were into single cells.
Vigorously pipette samples with 1 ml wide-bore pipette tips or if needed, regular tips.
Filter cells through MACS SmartStrainers (70um) and wash 1 ml HBSS (Ca2+/Mg2+ free) through the strainer.
Centrifuge at 500 x g, 4°C, 5 min to get rid of the supernatant.
Resuspend the cell pellet in 2 mL ACK Lysing buffer, and incubate on ice for 3 min to exclude RBCs.
Add 5 mL SS_buffer to stop ACK Lysing buffer reaction, then collect cells by centrifugation 500 x g, 4°C, 5 min.
Resuspend the pellet in 1ml of HBSS (Ca2+/Mg2+ free) (Gibco, 14170120).
Filter the cell suspension through Flowmi 40um cell strainers tube to remove debris and non-dissociated tissue fragments.
Collect cells by centrifugation 500 x g, 4°C, 5 min and proceed to Flex protocol.