Preparing a single cell or single nuclei suspension is a critical step for high throughput transcriptome and epigenome profiling. Our protocol provides a simple, reproducible, and universal method for single nuclei isolation. Contrary to other methods requiring detergent for nuclei isolation, our protocol is column-based, which is detergent and enzyme-free.
It enables nuclei isolation in less than 30 minutes. Our protocol simplifies nuclei isolation. It provides a robust method to prepare single nuclei suspension from hard-to-dissociate tissues.
To begin, pre-chill buffer A and B from a detergent-free nuclei isolation kit by placing them on ice for at least 30 minutes. Coat tubes and pipette tips with 5%BSA solution, which will enhance recovery of the nuclei. To coat the pipette tips, pipette 5%BSA solution two to three times and air dry them for two hours.
To coat the tubes, fill them with BSA and invert them three times. Remove the solution and air dry the tubes upside down on a clean tissue paper for two hours. Coat one collection tube and one 1.5 milliliter tube per sample.
Use forceps to gently break open the skull and remove soft tissues, skin, and bones from the ventral and dorsal sides of the skull. Then gently transfer the brain into a 30 millimeter dish containing ice cold PBS and mince the brain into small pieces using a razor blade. To isolate single nuclei, transfer the minced tissue to the filter cartridge provided in the nuclei isolation kit and remove excess solution and add 200 microliters of cold buffer A.Grind the tissue with a plastic rod for two minutes.
Add 300 microliters of cold buffer A to the filter cartridge and incubate it on ice with the cap open for 10 minutes. Then cap the cartridge and resuspend the tissue by inverting the tube five times. Centrifuge the sample tube at 16, 000 times g for 30 seconds to rupture the cells.
The flow-through contains intact nuclei, which form a colorless pellet at the bottom of the tube. Discard the filter and then resuspend the pellet by vortexing vigorously for 10 seconds. Pellet the nuclei again by centrifuging the solution at 500 times g for three minutes.
Then carefully discard the supernatant. Resuspend the pellet in 0.8 milliliters of cold buffer B by pipetting two to three times and centrifuge the nuclei at 600 times g for 10 minutes, which will separate them from the membrane debris. Carefully discard the supernatant.
Resuspend the isolated nuclei in 500 microliters of PBS with 5%BSA and keep the suspension on ice until FACS. To confirm nuclear morphology with HEX staining, transfer 100 microliters of the single nuclei suspension to a new tube using the BSA coated tips. Stain the nuclei by adding 0.1 microliter of HEX and gently vortexing the tube.
Transfer the nuclei suspension to a glass bottom dish and image of the nuclei using a fluorescence microscope with a laser excitation setting of 405 nanometers. Before performing FACS, filter the nuclei with a 40 micrometer cell strainer into a BSA coated tube. Dilute the filtered suspension by adding PBS with 5%BSA to a final volume of 1, 000 microliters.
Label two round bottom FACS tubes as control and stained and transfer 250 microliters of the nuclei suspension into the control tube using a BSA coated pipette tip. Transfer the remaining 750 microliters of the solution to the FACS tube labeled stained and add one microliter of HEX dye to stain the nuclei. Then mix it by slow vortexing.
Load the unstained control sample into the cell sorter and record 5, 000 events. Then load the stained sample and record 5, 000 events. Draw FACS gates for identification of single nuclei.
Nuclei can be selected by comparing the HEX fluorescent signal between the control and stained samples. Then sort HEX positive nuclei from the stained tube into a new 1.5 milliliter tube containing 50 microliters of PBS with 5%BSA. This protocol was used to generate single nucleus suspension directly from zebrafish brain tissue.
The isolated nuclei were stained with HEX and visualized with fluorescence microscopy. They appeared intact, round, and well-separated. Importantly, nuclear aggregation was absent.
Enrichment of isolated nuclei was performed with flow cytometry by gating on the presence of a HEX fluorescent signal. Unstained nuclei displayed background fluorescence while stained nuclei exhibited strong fluorescence signal. The unstained and stained nuclei were well-segregated in the violet channel.
While attempting this protocol, it's important to remember that single nuclei tends to stick on a plastic material. To prevent the potential loss of nuclei and increase efficiency, basic coating of the plastic material is highly recommended. Isolated single nuclei suspension can be further utilized for single nuclei RNA This technique elucidate cellular heterogeneity in complex biological systems and help define cell subtypes and gene networks.
Using a detergent and enzyme-free method registers the technical noise and bias introduced during sample preparation. This method has developed a simplified and logistical single nuclei isolation.
