GSM8941098: RTX tumor – CMO library; Mus musculus; RNA-Seq
Tumors were acutely dissected out, washed with 10ml PBS(Gibco), and then cut into small pieces with scissors. The tissues were dissociated with Tumor Dissociation Kit (Miltenyi Biotech). Briefly, tissues were digested at 37°C for 30 minutes in 2.35ml RPMI (Corning) with 100ml Enzyme D, 50 ml enzyme R, 12.5 ml  enzyme A, then passed through 70mm cell strainer. The cell suspension was centrifuged at 300g for 7 minutes at room temperature and supernatants were removed. The cell pellet was resuspended with 1mL Red Blood Cell Lysis Solution (Miltenyi Biotech) and incubated for 10 minutes at 4°C. 10mL DPBS with 1% BSA was used to stop the red blood cell lysis. Cells were centrifuged and resuspended in 5mL DPBS with 1% BSA. Dead cells were then removed by Dead Cell Removal Kit (Miltenyi Biotech) using MS columns (Miltenyi Biotech) and the MiniMACS magnetic separator. After that, live cells were isolated via centrifuging, incubated with 100ml unique Cell Multiplexing Oligo (CMO) mixture (10x Genomics) for 5 minutes at room temperature, and washed several times to remove excess CMOs. Cell viability was assessed via staining with Trypan Blue and a hemocytometer, and then diluted to achieve target concentration for 10X sequencing. Single-cell RNA-seq libraries were prepared according to 10x Genomics specifications (Single Cell 3′ Reagent Kits v3.1 User Guide CG000388, 10x Genomics, Pleasanton, CA, USA). Cellular suspensions (96% viable) at a concentration of 800 cells/µl, were loaded onto to the Chromium Controller to generate barcoded single-cell GEMs, targeting about 15000 single cells per sample. Barcoded and UMI labeled cDNA from poly A mRNA and DNA from the Cell Multiplexing Oligo tags (CMO) were generated simultaneously from the same cells inside the GEMs by incubation at 53 °C for 45 min, 85 °C for 5 min; held at 4 °C in a C1000 Touch Thermal cycler (Bio-Rad, Hercules). GEMs were broken, and cDNA and DNA from CMOs were amplified 11 cycles (98 °C for 3 min; 98 °C for 15 s, 63°C for 20 s) and the products separated by size selection with SPRIselect beads (Beckman Coulter, Indianapolis, IN). Gene expression libraries and CMOs libraries were generated independently. The fraction containing the cDNA was enzymatically fragmented, end repaired, A-tailed, and ligated to adaptors provided in the kit. A unique sample index for each library was introduced through 12 cycles of PCR amplification using the indexes provided in the kit (98 °C for 45 s; 98 °C for 20 s, 54 °C for 30 s, and 72 °C for 20 s). To obtain the CMO libraries, the fraction containing the CMO was PCR amplified with unique Indexes for 6 cycles using the same conditions as above. Quality and quantity of the libraries was assessed using an Agilent Bioanalyzer 2100 (Santa Clara, CA). Average cDNA size was 1600bp, average gene expression library 549bp and CMO library 212bp.