TITLE: JAX-Sen: Mouse heart dissociation for single-cell RNA sequencing
AUTHORS: Ramalakshmi Ramasamy, Juliana Alcoforado Diniz, Patrick Fleming, Jessica Garofalo, Paul Robson

[DESCRIPTION]
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.

[STEPS]
SECTION: Reagents and Materials
1. 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

 


SECTION: Procedure
2. Collect shipment from the warehouse.
SECTION: Procedure
2.1. Fill up ice buckets and prep them with reagents:
Bucket 1(small): PBS, HBSS, DMEM+FBS, SS_buffer
Bucket 2 (large): For mincing tissue
SECTION: Procedure
2.2. Confirm if the sample is cold. Take pictures.
SECTION: Procedure
2.3. Place tubes on ice and mince the tissue (<1mm) with sterile micro scissors.
SECTION: Procedure
2.4. After mincing, transfer the sample into labeled 5 ml tubes. 
SECTION: Procedure
2.5. Centrifuge at 500 x g, 4°C, 5 min to get rid of the supernatant.
SECTION: Procedure
2.6. Resuspend the pellet in ice-cold PBS (Ca+ and Mg+ free) and pipette up and down gently with wide-bore pipette tip.
SECTION: Procedure
2.7. Centrifuge at 500 x g, 4°C, 5 min to get rid of the supernatant. 
SECTION: Procedure
2.8. Resuspend the pellet in ice-cold PBS (Ca+ and Mg+ free) and pipette up and down gently with wide-bore pipette tip.
SECTION: Procedure
2.9. Centrifuge at 500 x g, 4°C, 5 min to get rid of the supernatant. 
SECTION: Procedure
2.10. 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.
SECTION: Procedure
2.11. Stop the digestion by adding an equal volume (1ml) of DMEM/10% FBS.
SECTION: Procedure
2.12. Centrifuge at 500 x g, 4°C, 5 min to get rid of the supernatant.
SECTION: Procedure
2.13. 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.
SECTION: Procedure
2.14. Vigorously pipette samples with 1 ml wide-bore pipette tips or if needed, regular tips.
SECTION: Procedure
2.15. Filter cells through MACS SmartStrainers (70um) and wash 1 ml HBSS (Ca2+/Mg2+ free) through the strainer.
SECTION: Procedure
2.16. Centrifuge at 500 x g, 4°C, 5 min to get rid of the supernatant.
SECTION: Procedure
2.17. Resuspend the cell pellet in 2 mL  ACK Lysing buffer, and incubate on ice for 3 min to exclude RBCs.
SECTION: Procedure
2.18. Add 5 mL SS_buffer  to stop  ACK Lysing buffer reaction, then collect cells by centrifugation 500 x g, 4°C, 5 min.
SECTION: Procedure
2.19. Resuspend the pellet in 1ml of HBSS (Ca2+/Mg2+ free) (Gibco, 14170120).
SECTION: Procedure
2.20. Filter the cell suspension through  Flowmi 40um cell strainers tube to remove debris and non-dissociated tissue fragments. 
SECTION: Procedure
2.21. Collect cells by centrifugation 500 x g, 4°C, 5 min and proceed to Flex protocol.