GSM6593314: B2m-tdTomato Positive; Mus musculus; RNA-Seq
Mice were perfused with HBSS without cations containing 5ug/ml actinomycin and 10uM triptolide. Brains were dissected out of skull, chopped into small pieces, then dissociated in EBSS buffer with papain (20U/ml), Dnase (100U/ml), actinomycin (5ug/ml), triptolide (10uM), anisomycin (27ug/ml), 22.5mM D-glucose, 0.5mM EDTA, 2.2g/L NaHCO3, and 5.5mM L-cysteine  at pH 7.4. Dissociations were done at 37c for 30 minutes with triturations every 10 minutes. Dissociated tissue was passed over 70um filters and myelin was removed with 2 rounds of debris removal solution (Miltenyi) following manufacturer's recommendation. Cells were labeled with Zombie Violet for dead cell exclusion then sorted on Aria IIu (VBR) into viable;tdTomato+ and viable;tdTomato- fractions. Libraries were constructed following manufacturer's instructions (Gene Expression 3' v3 chemistry, 10x genomics). For all 3 samples, cells were loaded at concentrations aiming for capture of 10,000 cells.