GONE in 360 seconds: gentleMACS Octo Dissociator-based Nuclei Extraction
Deepak Balamurali, Kinga M. Wrona, Lea Eich, Anika Witten, Alba M. Albert, Marc N. Hirt, Thomas Eschenhagen, Monika Stoll
Abstract
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
1. Pre-processing
Pre-cool the buffers, and consumables with sample contact (e.g. gentleMACS C Tube and SmartStrainers) at 4°C 0h 15m 0s. Similarly, place the gentleMACS Octo Coolers in -20°C .
Pre-cool the centrifuge to 4°C at the start of the experiments.
Per extraction, add RNase inhibitor (final concentration 0.2U/µL ) to pre-cooled 4mL Nuclei Extraction Buffer.
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), 7.2
In case of RNAbased downstream applications, add RNase inhibitor (final concentration 0.2U/µL ).
Retrieve fresh/frozen tissue 50-100mg and cut into small pieces using sterile forceps and a surgical blade.
2. Nuclei Extraction
Add 2mL ice-cold lysis buffer to each pre-cooled gentleMACS C Tube.
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.
3. Magnetic Labelling & Purification
Place previously cooled gentleMACS Octo Coolers on the C tubes to help maintain the temperature.
4. Quality Check/Control
Run gentleMACS Program 4C_nuclei_1 on the gentleMACS Octo Dissociator.
After termination of the program, detach C Tube from the gentleMACS Dissociator and place the C Tube immediately on ice.
Apply nuclei suspension to a MACS SmartStrainer (70 µm) placed on a 15 mL tube. Wash MACS SmartStrainer with 2mL ice-cold Nuclei Extraction Buffer.
Discard MACS SmartStrainer and centrifuge nuclei suspension at 300x g,4°C,0h 0m 0s for 0h 5m 0s. Carefully aspirate supernatant completely.
Resuspend nuclei pellet with ice-cold nuclei separation buffer slowly and gently pipetting the sample up and down 10 times.
Apply nuclei suspension to a MACS SmartStrainer (30 μm) placed on a 15 mL tube.
Collect nuclei suspension and proceed immediately with downstream applications (in this case, purification with Anti-Nucleus MicroBeads)
3. Magnetic Labelling & Purification
Determine the nucleus number using an automated Cell Counter or Neubauer chamber.
Centrifuge nucleus suspension at 300x g,4°C,0h 0m 0s for 0h 5m 0s. Pipette off supernatant completely.
Resuspend 1x 106 nuclei from the pellet in 450µL of nuclei separation buffer.
Add 50 µL of Anti-Nucleus MicroBeads to each reaction tube.
Mix well and incubate for 0h 15m 0s in the refrigerator (2-8°C).
Add 2mL nuclei separation buffer (independent from nucleus sample input) and proceed to magnetic separation.
Place the column in the magnetic field of a suitable MACS Separator.
Prepare the column by rinsing it with 3mL of nuclei separation buffer.
Apply the nuclei suspension onto the column. Collect flowthrough containing debris.
Wash the column twice with 1mL of nuclei separation buffer. Collect debris that passes through and combine with the flowthrough from the previous step.
Remove the column from the separator and place it on a suitable collection tube.
Pipette the appropriate amount of nuclei separation buffer onto the column depending on the nucleus sample input.
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.
4. Quality Control (optional)
Add DAPI Staining Solution (final concentration of 0.25 µg/ mL) to a small fraction of the nuclei suspension. Mix by inverting gently.
Incubate for 0h 5m 0s at 4°C.
Load sample on a hemocytometer or on a flow cytometer and analyze sample according to manufacturer’s recommendation.
