title: GSM7744846: Brain, conditioned and transplanted, GFP+ CD45+ CD11b+ cells; Mus musculus; RNA-Seq
library_construction_protocol: The brain was minced in ice-cold HBSS 1X (Thermo Fisher Scientific 14185052), the pieces were collected by centrifugation at 400g for 5 minutes and digested using the NTDK Neural Tissue Dissociation Kit (P) (Miltenyi Biotec 130-092-628) at 37° C for 30 minutes following the manufacturer's guidelines. Digested samples were quenched using ice-cold HBSS 1X and filtered through a 70 μm cell strainer, resuspended in 33% isotonic Percoll PLUS (GE Healthcare GE17-5445-01) and spun at 700 g for 15 minutes for myelin removal. The cell pellet was washed with FACS-BL buffer (PBS-1X supplemented with 5% Fetal Bovine Serum and 1% BSA fraction V) and kept on ice for FACS staining. Bone marrow cells (BM, from femurs and tibiae) were extracted in RPMI supplemented with 10% FBS, 4U/mL Heparin and 0.2U/mL Dnase I  and filtered using a 30 μm cell strainer and kept on ice for FACS staining. For FACS staining, cells were resuspended in 10% vol/vol Mouse BD Fc Block™ (clone 2.4G2 BD Biosciences) for 10 minutes and stained in the dark for 30 min using the following antibodies: anti-mouse CD45 PE-Cy7 (clone 104 Thermo Fisher Scientific), anti-mouse CD45 PE-Cy7 (clone 30F11 Thermo Fisher Scientific), anti-mouse/human CD11b-PE (clone M1/70 BioLegend), anti-mouse/human TER-119 PE-Cy5 (TER-119, BD Biosciences), anti-mouse Ly6C BV605 (clone AL-21 BD Biosciences), anti-mouse CD3 APC (clone 17A2 Thermo Fisher Scientific), anti-mouse CD19 PB (clone 6D3 BioLegend); eFluor™780 (Thermo Fisher Scientific 65-0865-18) was used as a viability dye. After staining, cells were washed and resuspended in FACS-BL buffer. Stained cells were sorted using a MA900 Multi-Application Cell Sorter (Sony Biotechnology) flow cytometer in cold FACS-BL buffer. Cells were processed using the Chromium Controller and the Chromium Next GEM Single Cell 3' Reagent Kits (10x Genomics). The libraries were sequenced via Illumina Novaseq 6000 sequencer.