GSM9352250: 1E NSG-CSF1 Without iMGL engrafted; Mus musculus; RNA-Seq
Mice were perfused with ice-cold 1X HBSS with 10 U/ml heparin, together with transcriptional inhibitors were added actinomycin D (5 µg/ml), triptolide (10 µM) as was done in other studies 12. Samples were collected on ice in 1X HBSS that contained anisomycin (27.1 µg/ml) as a third transcription inhibitor, as well as RNase Inhibitor (0.2 U/μl) and DTT (1 mM). All compounds were used in all subsequent solutions at the same concentrations, together with and sample were kept at 4°C throughout the process. Brain dissociations were performed based on an adapted protocol previously described for CyTOF tissue processing. Brains were dissociated using a papain-based dissociation kit at 37 C and 5% CO2 for 30 min (according to the manufacturer's instructions) with gentle titurations using 10 and 5 ml serological pipettes in 10 min intervals. Papain was inhibited by albumin-ovomucoid solution mixed with DNAse. Single cell suspensions were filtered through a 40 µm cell strainer and resuspended in Myelin Removal Beads II solution according to manufacturer's instructions. Dynamag magnets were used to remove myelin from the solution. For cell culture cells, cells were detached using 37°C PBS and a cell scraper. After passing through a 40 µm cell strainer, cell suspensions were spun at 300 g for 5 min and resuspended in 1X PBS with 0.1% before loading on the Chromium device. 10X single-cell libraries were prepared following the manufacturer's guidelines per mouse, without pooling. As 10X chips are limited to 8 samples per run, some experiments were run in multiple processing batches.