Aug 30, 2024
JAX-Sen: Mouse kidney 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;
- 22Department 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 2024. JAX-Sen: Mouse kidney dissociation for single-cell RNA sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk833wl5r/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: 102773
Keywords: JAX-Sen
Funders Acknowledgement:
National Institute on Aging (NIA) JAX-Sen Senescence Tissue Mapping Center
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 kidney. We compare young (6 months old) and old (24 months old) mouse kidneys using scRNAsequencing. This protocol describes the single-cell dissociation of the kidneys 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
- GentleMACS dissociator
- GentleMACS C tubes
- DMEM + 10% FBS
- ACK lysis buffer – 2 ml/sample
- SS_buffer– 5 ml/sample
- 40um cell strainer
- 70uM 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): Enzyme mix, PBS, SS_buffer
Bucket 2 (large): For mincing tissues and sample tubes
Confirm if the sample is cold. Take pictures. Transfer sample to 5 ml tubes.
Prepare enzyme mix by adding 2.35 mL of serum-free RPMI 1640 or DMEM, 100 µL of Enzyme D, 50 µL of Enzyme R, and 12.5 µL of Enzyme A of the Multi Tissue Dissociation Kit 1 into a gentleMACS C Tube for up to 0.5 g of tissue.
Mince mouse kidneys (2-4 mm3) with sterile micro scissors.
After cutting, transfer the sample into labeled 5 ml tubes.
Tightly close C Tube and attach it upside down onto the sleeve of the gentleMACS Dissociator.
Run the gentleMACS Program Multi_B. If using the heating function of the gentleMACS Octo Dissociator with Heaters run program 37C_Multi_E.
After termination of the program, detach C Tube from the gentleMACS Dissociator.
Resuspend sample and apply the cell suspension to a MACS‱ SmartStrainer (70 µm) placed on a 5 mL tube.
Wash MACS SmartStrainer (70 µm) with 4 mL of ice-cold PBS.
Centrifuge at 300 x g, 4°C, 7 min to get rid of the supernatant. Aspirate 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 ice cold PBS.
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.