GSM7881870: E15.5, En1/2 CKO cerebellum, replicate 2; Mus musculus; RNA-Seq
Animals were dissected in ice cold Hank's balanced salt solution (HBSS) (Gibco) and were pooled together for downstream steps. Two replicate experiments were performed. On ice, cerebella were detached from the rest of the brain using forceps and dissociated in Accutase (Innovative Cell Technologies) at 37°C for 7-10 minutes. Following dissociation, accutase was washed out using neural stem cell (NSC) media (Neurobasal, supplemented with N2, B27 and non-essential amino acids, Gibco). Samples were triturated by pipetting up and down using a P1000, filtered through a 35um cell strainer (Falcon)and then centrifuged at 500g at 4°C for 5 minutes. Cells were washed again in NSC media and were finally filtered into polypropylene round bottom tubes (Falcon) prior to sorting for tdT+ cells. tdT+ cells were sorted into polypropylene round bottom tubes, spun down at 500g at 4°C for 5 minutes and resuspended in NSC media prior to single cell library preparation. Single cell libraries were prepared using 10X Genomics' Chromium Next GEM Single Cell 3' Reagent Kits, v3.1 according to manufacturer's instructions. Prior to loading the 10X Genomics chip, the limited sample preparation protocol was used. Cell viability was quantified using 0.2% (w/v) Trypan Blue on a hemocytometer, and 10,000 viable cells were targeted per pooled sample.