The main objective of this protocol is to study the interaction of genetic and environmental factors in the context of heart development and their contribution to congenital heart defects. Single nuclei approaches, such as single nuclei RNA-Seq and single nuclei ataxic, are emerging as key methods to study cellular heterogeneity in health and disease during heart development. One limiting step of these experiments is that all the sample have to be run simultaneously.
While working with all the experimental conditions, collecting fresh enough material is for all the condition simultaneously could be challenging. Although there have been significant progress in the field in single cell for recent years, the main difficulty is processing fresh samples. Avoiding the difficulty is extremely benefit to identify the molecular mechanism underlying congenital heart disease defect.
This protocol describes the steps to follow for successfully isolating high-quality nuclei from frozen cardiac cell suspension obtained from mouse embryos, and our protocol is compatible with downstream single nuclei RNA-Seq and single nuclei ataxic. We hope that this procedure will help interested researchers and encourage them to use this powerful method for their research. Begin by placing the embryos dissected from the euthanized two to six month old pregnant female mouse in a cold complete medium with 1%fetal bovine serum.
After removing the endometrial tissue, placenta, and yolk sac from the embryo under 5X magnification, dissect the cardiac progenitors from the embryonic heart region using forceps. Pull all the heart regions dissected from five mouse embryos in a 1.5 milliliter micro centrifuge tube and centrifuge them in a swinging bucket pre-chilled to four degrees Celsius. Remove the supernatant and wash the tissue with one milliliter of tissue culture grade PBS.
Centrifuge the tubes at 300G for one minute at four degrees Celsius. Then remove the supernatant and repeat two washes with one milliliter PBS. Once done, replace the PBS with 0.05%trypsin EDTA.
Centrifuge and repeat two washes with 0.05%trypsin EDTA. To digest the tissue, add 50 microliters of 0.05%trypsin EDTA and heat for 10 minutes at 37 degrees Celsius. Next, apply a gentle mechanical dissociation after five minutes by aspirating the suspension up and down using a wide orifice filter tip.
Inactivate the trypsin digestion activity by adding 350 microliters of complete medium to the dissociated cells. Pass the cell suspension through a 40 micrometer cell strainer. Prepare the cells for freezing by centrifuging the freshly dissociated cell suspension.
Carefully remove the supernatant and resuspend the pellet in one milliliter of chilled freezing medium. Transfer the cell suspension into a pre-cooled cryo vial and place the cryo vial in a pre-cooled freezing container. Place the freezing container at minus 80 degrees Celsius for four to eight hours before transferring it to liquid nitrogen for sequencing experiments.
Begin by placing the frozen dissociated embryonic heart cell suspension containing cryo vials in a 37 degrees Celsius water bath for one to two minutes. Add one milliliter of complete medium pre-warmed at 37 degrees Celsius to the thawed suspension, and mix it three times with a pipette. Then, transfer the suspension to a 15 milliliter conical tube containing 10 millimeters of warm complete medium.
Pass the entire cell suspension over a 30 micrometer strainer and rinse the strainer with one to two milliliters of warm complete medium. Collect the flow-through in the same conical tube. Next, centrifuge the tubes at 300G for five minutes.
After removing the supernatant, wash the pellet with PBS and transfer the cell suspension into a 1.5 milliliter micro tube. Centrifuge the cell suspension and add 100 microliters of the provided magnetic microbeads to the pelleted cells. Homogenize the suspension five times using a wide bore pipette tip before 15 minutes of incubation at room temperature.
In the meantime, rinse the magnetic separation column with 500 microliters of binding buffer. Once the incubation is complete, dilute the microbead cell suspension mixture using 500 microliters of binding buffer. Next, add 0.6 milliliters of the cell suspension to the magnetic separation column.
Collect the live cell effluent in a 15 milliliter centrifuge tube. Next, rinse the 1.5 milliliter micro tube that contained the cell suspension using one milliliter of binding buffer. After rinsing, transfer the binding buffer from the 1.5 milliliter micro tube to the column, and collect the effluent in the same 15 milliliter tube.
Repeat the rinsing of the 1.5 milliliter tube using one milliliter of binding buffer. After centrifuging the effluent at 300G for five minutes, remove the supernatant without disrupting the cell pellet. Add one milliliter of the PBS BSA solution and gently mix by pipetting using a wide orifice pipette tip.
Then, transfer the cell suspension into a 1.5 milliliter micro tube. Again, centrifuge the cell suspension and remove the supernatant without disrupting the cell pellet. Repeat the two washes of one milliliter of PBS BSA.
After removing the one milliliter of PBS BSA, resuspend the cells in 100 microliters of PBS BSA solution. Mix it by pipetting 10 times. Determine the concentration by performing a trypan blue assay to ensure a cell count of more than or equal to 100, 000 cells, and perform a fluorescence-based assessment for the viability of the cells.
The additional step of sorting and removing the dead cells after thawing allowed the procurement of a cleaner cell suspension with significantly higher cell viability and improved the quality of the starting sample. For more accuracy, two different counting methods were used to quantify the cells and nuclei, including trypan blue and the fluorescence-based assessment of the viability. In this protocol, it was observed that the cell viability passed from 80 to 90%before nuclei isolation to less than 5%after nuclei isolation.
To begin, clean the working place and materials with 70%ethanol and RNase removing solution. Freshly prepare lysis buffer, lysis dilution buffer, 0.1X lysis buffer, and wash buffer, and maintain the buffers on ice. Pre-chill swinging bucket centrifuges to four degrees Celsius.
Centrifuge the cardiac progenitor cell suspension in a swinging bucket centrifuge. Gently remove all the supernatant without touching the bottom of the tube. Add 100 microliters of chilled 0.1X lysis buffer to the cell suspension, and mix it by pipetting 10 times.
After five minutes of incubation on ice, add one milliliter of chilled wash buffer and mix it. After centrifuging the suspension, remove the supernatant and repeat two more washes with chilled wash buffer. Prepare the diluted nuclei buffer.
After resuspending the nuclei in the nuclei buffer, place the nuclei stock solution on ice. When the quality of the isolated nuclei was assessed by the evaluation of the nuclear membrane morphology, the isolated nuclei appeared smooth and uniformly round under brightfield microscopy, indicating an effective isolation process.
