Isolation of Single Cells from Human Brain Organoids for Single-Cell RNA Sequencing

To begin, collect up to five human brain organoids generated from induced pluripotent stem cells in one well of a six well plate. Aspirate excess culture media, and wash the organoids once with PBS. Using a scalpel, mince the organoids thoroughly.

Add enzyme mix one to the minced organoids, and incubate at 37 degrees Celsius with 20%oxygen and 5%carbon dioxide at 90 RPM for 10 to 15 minutes. Mix the organoid suspension using a 1000 microliter pipette tip. Then add enzyme mix two, and incubate at 37 degrees Celsius for 10 to 15 minutes.

Stop dissociation by adding 10 milliliters of stop solution. Filter the cell suspension on a 70-micron cell strainer. Centrifuge the filtered cell suspension at 300 G for 10 minutes at four degrees Celsius.

Aspirate the supernatant, and re-suspend the pellet in four milliliters of cold 0.04%BSA. Now filter the cell suspension on a 40-micron cell strainer, and count the cells on a cell counter. Centrifuge the required amount of cell suspension, aspirate the supernatant, and re-suspend the pellet in NSB plus medium according to a microfluidics-based scRNA-Seq kit manual.