Apr 28, 2025
JAX-Sen: Mouse placenta single-cell dissociation for single-cell RNA sequencing
- Ramalakshmi Ramasamy1,
- Juliana Alcoforado Diniz1,
- Jessica Garofalo1,
- Patrick Fleming1,
- 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, Jessica Garofalo, Patrick Fleming, Paul Robson 2025. JAX-Sen: Mouse placenta single-cell dissociation for single-cell RNA sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9korml3e/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 26, 2025
Last Modified: April 28, 2025
Protocol Integer ID: 174722
Funders Acknowledgements:
National Institute on Aging (NIA) JAX-Sen Senescence Tissue Mapping Center Grant
Grant 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. This protocol describes the single-cell dissociation of the placenta tissue before library preparation and sequencing.
Reagents and Materials:
Reagents and Materials:
1. 6 cm Petri dishes
2. Ice-cold MACS buffer
3. 5 ml Protein lobind tubes
4. Micro scissors/ scalpel
5. Ice-cold PBS
6. Wide-bore pipette tips
7. TrypLE express (10 volumes)
8. Dispase (0.4 g/ml)
9. Collagenase
10. DMEM + 10% FBS
11. ACK lysis buffer
12. CS_buffer
13. 40 um cell strainer (Flowmi)
14. 70 uM strainer
Procedure:
Procedure:
Add sample labels and collect shipment from the warehouse.
Procedure:
Procedure:
Fill up ice buckets and prep them with reagents:
Bucket 1: PBS, MACS buffer, DMEM, DMEM+FBS, SS_buffer
Bucket 2: For mincing tissue, either in 6 cm dishes or in tubes – protocol A
Bucket 3: For mincing tissue, either in 6 cm dishes or in tubes – protocol B
Bucket 4: extra ice
Confirm if the sample is cold.
Notes:
Optional: Place the sample on a dish with MACS buffer and take pictures.
Place tubes on ice and mince the tissue (<1mm) with sterile micro scissors (or) mince
using a scalpel on 6 cm dish w/MACS buffer on ice.
After mincing, transfer the sample into labeled 5 ml/ 10 ml tubes.
Centrifuge at 800 x g, 4°C, 5 min to get rid of the supernatant.
Resuspend the pellet in ice-cold PBS and pipette up and down gently with wide bore
pipette tip.
Centrifuge at 800 x g, 4°C, 5 min to get rid of the supernatant.
Resuspend the pellet in ice-cold PBS and pipette up and down gently with wide bore
pipette tip.
Centrifuge at 800 x g, 4°C, 5 min to get rid of the supernatant.
Add 10 volumes (~500 uL) of [1937.5 uL TrypLE + 50 uL Dispase + 12.5 uL RNAse
inhibitor + 250 uL DNAseI] to the pellet and enzymatically digest at 4°C for 35 min
with slow rocking.
Check every 5-10 mins minutes for dissociation, under the microscope.
Stop the digestion by adding an equal volume (~500 uL) of DMEM/10% FBS.
Filter cells through MACS SmartStrainers (70um)
Wash 1.5 ml DMEM through the strainer. -----------à this is the filtrate cell suspension
Filtrate: Leave on ice.
Residue:
- Collect the residue with wide bore pipette tips and transfer them to a new 2 ml
conical tube.
2. Add collagenase Type IV (0.5 mg/mL) and DNase I (0.2 mg/mL) to the residue and
enzymatically digest for 10 min at 37 °C.
[500 ul DNaseI + 1.25 mg collagenase + 12.5 uL RNase inhibitor + 1862.5 uL DPBS
(make up to 2.5 ml)]
3. Filter cells through MACS SmartStrainers (70um)
4. Wash 1.5 ml DMEM through the strainer. ---> this is the residue cell
suspension
Pool the filtrate and residue cell suspensions and proceed to step 16
Centrifuge at 800 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 CS_buffer to stop ACK Lysing buffer reaction, then collect cells by
centrifugation 800 x g, 4°C, 5 min.
Resuspend the pellet in 1ml of CS_buffer.
Filter the cell suspension through 40um cell strainers tube to remove debris and non-
dissociated tissue fragments.
Collect cells by centrifugation 800 x g, 4°C, 5 min and proceed to 10X Flex protocol/ cell
sorting.
CS_buffer (Cell suspension 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 | 2mM | 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 |