Isolation of Single Nuclei from Human Brain Organoids for Single-Nucleus RNA Sequencing

To begin, transfer four human brain organoids generated from induced pluripotent stem cells into a six-well plate containing chilled PBS. Cut each organoid into four pieces. Using scissors, cut the tip of a 1, 000 microliter pipette tip and use it to transfer organoid pieces into a two milliliter douncer.

Aspirate PBS completely and add one milliliter of NP-40 lysis buffer. Dounce the organoids three times with dounce pestle A followed by pestle B.Transfer the suspension into a 15 milliliter centrifuge tube. Wash the douncer with one milliliter of lysis buffer and transfer the solution to a centrifuge tube.

After five minutes of incubation at room temperature, centrifuge the organoid suspension at 500 G for five minutes. Aspirate the supernatant leaving 50 microliters in the tube. Add one milliliter of NSB Plus medium into the tube.

And after five minutes, mix to resuspend the pellet. After preparing a Percoll gradient in the tube, layer the organoid suspension on top of it. Next, centrifuge the layered organoid suspension at 500 G for five minutes at four degrees Celsius.

Aspirate the supernatant and resuspend the pellet in NSB Plus medium. Filter the nuclei solution on a 40 micron cell strainer. Then, stain the nuclei using DAPI and count them in a Neubauer chamber.

Centrifuge the required amount of nuclei solution and resuspend the pellet in NSB Plus medium according to a microfluidics based snRNA-seq kit manual.