Isolation of Nuclei from Fresh Frozen Glioma Tissues: A Method to Obtain Intact Nuclei from Glioma Tumor Samples
Transkript
To isolate nuclei, begin by taking a fresh frozen glioma sample – a tumor that originates in the glial cells of the brain – in a Petri dish.
Place the petri dish on ice to avoid tissue degradation.
Next, mince the tissue sample into small pieces.
Add a pre-chilled lysis buffer to the minced tissue pieces. Transfer the mixture to a Dounce homogenizer. Using the pestle, homogenize the tissue to disintegrate the cells and initiate lysis.
Now, transfer the homogenate to a microcentrifuge tube containing a fresh lysis buffer.
Mechanically disrupt the homogenate using a wide-bore pipette tip. This step facilitates the release of cytoplasmic organelles from the cells.
Subsequently, using an appropriately sized cell strainer, filter the homogenate into a fresh tube to remove tissue debris and other contaminants.
After filtration, take the filtrate containing various cell organelles.
Centrifuge the filtrate at a low speed to pellet the denser nuclei at the bottom while the other cell organelles remain in the supernatant.
Discard the organelle-containing supernatant. Store the crude nuclei-rich pellet in a suitable storage buffer for further downstream sequencing.
