GSM9171621: 8803 3; Mus musculus; RNA-Seq
At designated time points, tumor-bearing mice were euthanized and brains collected following cardiac perfusion and chopped up using a scalpel, dissociated, and suspended using a mixture of IMDM 1X (Corning) containing 2% inactivated FBS (Millipore Sigma), collagenase (100 μg/mL) and DNase (20 units/mL). Samples were incubated for 35–40 minutes at 37°C with agitation. The tissue was filtered through a 70 μm nylon cell strainer (BD Biosciences) and then underwent centrifugation at 4°C. Half of the pellet representing the whole TME was resuspended in culture media, and the other half in a 20 mL mixture of 5.4 mL Percoll Plus (GE Healthcare) overlaid with 12 mL 1X PBS and 0.6 mL 10X PBS (Corning) for immune enrichment. The tube was centrifuged at 800g for 10 minutes at 4°C, with 9 acceleration and 0 deceleration. After centrifugation, the immune-enriched cell pellet was collected, washed, mixed with the whole TME samples at a ratio of 1:1, stained with Trypan blue dye (MilliporeSigma), and counted using a Countess II FL automated cell counter in a Countess cell-counting chamber (Invitrogen, Thermo Fisher Scientific). Done through standard protocol by NuSeq Core (Jennifer Chai)