GSM5239409: Met_s2 [MULTI-seq barcodes]; Homo sapiens; RNA-Seq
At 10 weeks old, MITRG mice were injected intracardially with 500,000 mCherry labeled 231BR cells as previously described. 25 days after intracardiac injection and following perfusion with ice cold PBS containing 5μg/ml actinomycin D (act D), whole metastatic brains were briefly imaged on a dissection microscope (Leica Biosystems, DMC 2900) for mCherry and GFP intensity.  Half brains were then dissected, fixing the left hemisphere in 4% PFA for histology and the right hemisphere was prepped for dissociation as described in Hasselmann and Coburn et al. (2019) with  modifications. The cerebellum was removed and the whole right hemisphere was stored briefly in RPMI 1640 containing 5μg/mL act D, 10μM triptolide, and 27.1ug/mL anisomycin. Tissue dissociation was then performed using the Tumor Dissociation kit, human (Miltenyi Biotec) and the gentleMACS OctoDissociator with heaters (Miltenyi Biotec) according to manufacturer guidelines with modifications. Briefly, tissue was cut into ~1mm pieces and placed into the C-tubes with the kit's enzymes, 5μg/mL act D, 10μM triptolide, and 27.1ug/mL anisomycin and samples were dissociated using the preprogrammed soft tumor protocol. Following enzymatic digestion, samples were strained through a 70μm filter and pelleted by centrifugation. Myelin and debris were removed by resuspending the pellet in 8mL 23% Percoll, overlaid with 2mL of 1X DPBS, spinning at 400xg for 25 minutes at 4°C, with acceleration and brake set to 0, and discarding the myelin band and supernatant.  For barcoding of cells from each individual mouse the MULTI-seq lipid- tagged indices for sample multiplexing for scRNAseq protocol was followed (McGinnis et al, 2019). Lipid anchor and co-anchor reagents were a generous gift from Zev Gartner, and barcode index oligos were purchased from Integrated DNA Technologies, Inc. Cells were resuspended and washed with 15 mL cold DPBS and pelleted by centrifugation (10 minutes, 400xg). The supernatant was discarded, and cells were resuspended in 180μL of DPBS. 20μL of 20μM Anchor:Barcode solution was added to a final concentration of 2μM, and incubated on ice for 5 minutes. Next 20μL of 20μM Co-Anchor solution was added, gently mixed and incubated for 5 minutes. After incubation 1mL 1% BSA in DPBS was added and cells were pelleted by centrifugation (5 minutes, 400xg). Finally, the supernatant was removed and washed a second time with 1mL 1% BSA in PBS and pelleted by centrifugation (5 minutes, 400xg). Next, mouse cell removal was performed by resuspending cell pellets in 160μL FACS buffer (0.5% BSA in 1X DPBS) + 40μL Mouse cell removal beads (Miltenyi Biotec) and incubated at 4°C for 15 minutes. Mouse and human cells were then separated using LS columns and the MidiMACs separator (Miltenyi Biotec) and the human cells were collected in the flow through. Human cells were pelleted via centrifugation (10 minutes, 400xg) and control samples and metastatic samples were then pooled separately. Cells were resuspended to ~1,000 cells per microliter in FACS buffer, according to counts performed on a hemocytometer. MULTI-seq tagged isolated human cells were pelleted via centrifugation for 10 minutes at 400xg and resuspended to approximately 1,000 cells per microliter in FACS buffer. Replicate cell pools were counted by hemocytometer and pooled for further single cell sequencing. Final cell suspensions were counted on the Countess II automated cell counter to determine actual concentration for droplet generation. Cells were loaded onto the 10x Genomics Chromium Single Cell Gene Expression 3' v3 Chemistry kits for GEMs generation. Following the Chromium Single Cell 3′ Reagents Kits version 3 user guide (CG000183 Rev C), cells were loaded to achieve approximately 10,000 cells for capture. MULTI-seq barcode libraries were prepared according to McGinnis et al. 2019 MULTI-seq protocol.  Libraries were sequenced on the Illumina NovaSeq 6000 platform to achieve an average read depth of 50,000 mean reads per cell for 3' gene expression libraries. MULTI-seq barcode libraries were sequenced to achieve at least 5,000 reads per cell.