TITLE: GONE in 360 seconds: gentleMACS Octo Dissociator-based Nuclei Extraction
AUTHORS: Deepak Balamurali, Kinga M. Wrona, Lea Eich, Anika Witten, Alba M. Albert, Marc N. Hirt, Thomas Eschenhagen, Monika Stoll

[DESCRIPTION]
Single-cell and single-nuclei approaches are being extensively used to decipher the cellular heterogeneity of samples and to understand the genetic factors leading to various diseases. While single-cell RNA sequencing is quite straightforward, single-nuclei sequencing requires careful consideration in the isolation & processing of intact nuclei after dissociation. Numerous protocols have been developed for this purpose, but the size and morphology of cardiomyocytes make the process quite complicated.1-3 

Previously described processes also included steps involving manual homogenization using a Dounce homogenizer and Fluorescence-activated Cell Sorting.2,4 These steps have an increased propensity to vary from user to user and also produce stress on the cells, which further alters the transcriptional profile of these nuclei. 

To this end, we devised a simple direct protocol for processing cardiac tissue into intact nuclei for downstream single-nuclei RNA sequencing using the gentleMACS Octo Dissociator. This protocol has been extensively tested with Engineered Heart Tissues (EHTs) and also works well with frozen or fresh cardiac tissues from human biopsies.

[BEFORE_START]
All samples and reagents are kept on ice or at 4 °C at all steps of the protocol.
Prepare all buffers and reagents as described in the "Pre-processing" section.

[STEPS]
SECTION: 1. Pre-processing
1.  Pre-cool the buffers, and consumables with sample contact (e.g. gentleMACS C Tube and SmartStrainers) at 4 °C 15 min. Similarly, place the gentleMACS Octo Coolers in -20 °C  .
SECTION: 2. Nuclei Extraction
6. Add 2 mL  ice-cold lysis buffer to each pre-cooled gentleMACS C Tube. 


SECTION: 4.	Quality Check/Control
9. 
SECTION: 3.	Magnetic Labelling &amp; Purification
8. 
SECTION: 1. Pre-processing
3. Per extraction, add RNase inhibitor (final concentration 0.2 U/µL ) to pre-cooled 4 mL  Nuclei Extraction Buffer.
SECTION: 1. Pre-processing
4. Prepare the nuclei separation buffer (for resuspension) before further enrichment of the nuclei using Anti-Nucleus MicroBeads. Dilute as specified below:

MACS BSA Stock Solution 1:250 (0.04% final concentration) and
Nuclei Extraction Buffer 1:7 (14% final concentration) in phosphate-buffered saline (PBS), pH 7.2  

In case of RNAbased downstream applications, add RNase inhibitor (final concentration 0.2 U/µL ).

 


SECTION: 1. Pre-processing
5.  Retrieve fresh/frozen tissue   50-100 mg and cut into small pieces using sterile forceps and a surgical blade.  

 

7. Transfer tissue pieces to the gentleMACS C Tube containing Nuclei Extraction Buffer and directly proceed with the following steps until samples are dissociated. Close the gentleMACS C Tube and place it on the gentleMACS Dissociator. 
 

SECTION: 2. Nuclei Extraction
7. 
8. Place previously cooled gentleMACS Octo Coolers on the C tubes to help maintain the temperature.
9. Run gentleMACS Program 4C_nuclei_1 on the gentleMACS Octo Dissociator.
10. After termination of the program, detach C Tube from the gentleMACS Dissociator and place the C Tube immediately on ice.
11. Apply nuclei suspension to a MACS SmartStrainer (70 µm) placed on a 15 mL tube. Wash MACS SmartStrainer with 2 mL  ice-cold Nuclei Extraction Buffer.



12. Discard MACS SmartStrainer and centrifuge nuclei suspension at 300 x g, 4 °C  for 5 min. Carefully aspirate supernatant completely.

13. Resuspend nuclei pellet with ice-cold nuclei separation buffer slowly and gently pipetting the sample up and down 10 times.

14. Apply nuclei suspension to a MACS SmartStrainer (30 μm) placed on a 15 mL tube.

15. Collect nuclei suspension and proceed immediately with downstream applications (in this case, purification with Anti-Nucleus MicroBeads)
SECTION: 3. Magnetic Labelling &amp; Purification
16. Determine the nucleus number using an automated Cell Counter or Neubauer chamber. 
SECTION: 3. Magnetic Labelling &amp; Purification
17. Centrifuge nucleus suspension at 300 x g, 4 °C  for 5 min. Pipette off supernatant completely.

 

SECTION: 3. Magnetic Labelling &amp; Purification
18. Resuspend 1x 106 nuclei from the pellet in 450 µL  of nuclei separation buffer. 
SECTION: 3. Magnetic Labelling &amp; Purification
19. Add 50 µL of Anti-Nucleus MicroBeads to each reaction tube.
SECTION: 3. Magnetic Labelling &amp; Purification
20. Mix well and incubate for 15 min in the refrigerator (2-8 °C).

SECTION: 3. Magnetic Labelling &amp; Purification
21. Add 2 mL  nuclei separation buffer (independent from nucleus sample input) and proceed to magnetic separation.

SECTION: 3. Magnetic Labelling &amp; Purification
22. Place the column in the magnetic field of a suitable MACS Separator.
 

SECTION: 3. Magnetic Labelling &amp; Purification
23. Prepare the column by rinsing it with 3 mL  of nuclei separation buffer.

SECTION: 3. Magnetic Labelling &amp; Purification
24. Apply the nuclei suspension onto the column. Collect flowthrough containing debris.
SECTION: 3. Magnetic Labelling &amp; Purification
25. Wash the column twice with 1 mL of nuclei separation buffer. Collect debris that passes through and combine with the flowthrough from the previous step.
SECTION: 3. Magnetic Labelling &amp; Purification
26. Remove the column from the separator and place it on a suitable collection tube.
SECTION: 3. Magnetic Labelling &amp; Purification
27. Pipette the appropriate amount of nuclei separation buffer onto the column depending on the nucleus sample input.
SECTION: 3. Magnetic Labelling &amp; Purification
28. Immediately flush out the magnetically labeled nuclei by firmly pushing the plunger into the column. The nuclei extracted can directly be proceeded with for downstream applications such as single-nuclei RNA sequencing. 
SECTION: 4. Quality Control (optional)
29. Add DAPI Staining Solution (final concentration of 0.25 µg/ mL) to a small fraction of the nuclei suspension. Mix by inverting gently.
SECTION: 4. Quality Control (optional)
30. Incubate for 5 min at 4 °C.

SECTION: 4. Quality Control (optional)
31. Load sample on a hemocytometer or on a flow cytometer and analyze sample according to manufacturer’s recommendation. 
SECTION: 1. Pre-processing
2. Pre-cool the centrifuge to 4 °C at the start of the experiments.