GSM8138713: Brain, conditioned and transplanted, GFP+ CD45+ CD11b+ cells (n1, n2, n3); Mus musculus; RNA-Seq
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 fluorophore-tagged 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.   Cell Hashing, using oligo-tagged antibodies directed to mouse CD45 (clone 30-F11) and mouse MHCI (clone M1/42), was used to uniquely label cells from distinct mice. The sequencing of each mouse-specific tag alongside the cellular transcriptome allowed us to pool cells from three different mice per sample and prevent sample-specific batch effects that occur during single-cell RNA sequencing. Each unique oligo-tagged antibody mix was added to the flow cytometry antibody mix described above (0.15 µL/1x106 cells). 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. The unique oligo-tagged antibodies were as follows: MGLC, host MG, BM-CD11b+ mouse 1 (TotalSeqTM-B 0301 anti-mouse Hashtag 1 Antibody, BioLegend 155831), mouse 2 (TotalSeqTM-B 0302 anti-mouse Hashtag 2 Antibody, BioLegend 155833), mouse 3 (TotalSeqTM-B 0303 anti-mouse Hashtag 3 Antibody, BioLegend 155835); Naïve MG mouse 1 (TotalSeqTM-B 0301 anti-mouse Hashtag 1 Antibody, BioLegend 155831), mouse 2 (TotalSeqTM-B 0302 anti-mouse Hashtag 2 Antibody, BioLegend 155833), mouse 3 (TotalSeqTM-B 0303 anti-mouse Hashtag 3 Antibody, BioLegend 155835). 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.