GSM8108141: NOTCH3-WT, NOTCH3-R170C, WT6m_brain_male (2 replicates each) and mcao3d; Mus musculus; RNA-Seq
We used scissors to cut the whole brain into small pieces as much as possible, added 1ml mixed enzyme solution, transfered to a shaker and digested at 200rpm, 37 ° C for 20 minutes. Then we epeatedly blowed the digested tissue homogenate 10 times using a 1ml tip, and transfered the tissue homogenate to the cell filter. The cell filters were rinsed with 6ml of 0.15M physiological saline, then rinsed with 3ml of 100% Percoll to thoroughly remove any remaining brain cells on the filter. The cells were gently mix, and then slowly transfered to a 15ml centrifuge tube containing 2ml of 70% Percoll. Brain cells were centrifuged according to the following parameters: 800g, 18 ° C, increase speed by 3, decrease speed by 3, for 15 minutes. The upper myelin sheath was removed and the liquid is transferred to a clean and sterile 50ml centrifuge tube. After filling with PBS, brain cells were centrifuged at 700g, 4 ° C for 8 minutes. The precipitate obtained was the brain tissue single-cell suspension. Perform cell viability testing on the obtained single-cell suspension, using samples with cell viability greater than 80% and a cell count of 5 * 105 for subsequent experiments. Single-cell suspensions were loaded onto the Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit (10X Genomics, 1000414). Sample processing and library preparation was performed according to manufacturer's instructions.