GSM7610692: RAG D10 Gene Expression; Mus musculus; RNA-Seq
Whole dissected brains were chopped into 8 pieces of equal size and placed into C tube (Miltenyi Biotec, Cat#130-093-237) 1mg/mL Collagenase D (Millipore Sigma Cat#11213857001)  Brain tissue was digested using gentleMACS Octo Dissociator with heaters operating the 37°C Adult adult brain dissociation protocol for 30 minutes. After digestion, the cell suspension was strained over a sterile 70μm strainer (Fisher Scientific, Cat#22363548) and washed with 5mL FACS buffer containing ice cold DMEM/F12, 50mM HEPES, and 2% BSA. After removal of myelin by density centrifugation, the cell pellet was washed and remaining red blood cells were lysed with red blood cell lysis buffer. Cells were then re-suspended in FACS buffer and blocked with anti-CD16/32 for 15 minutes on ice. Next, cells were stained with fluorescent antibodies on ice for 15 minutes shielded from light. C57BL/6 and RAG1-KO cells from individual mice were labeled using a CellPlex multiplexed oligo labeling kit in accordance with the manufacturer's protocol (10x Genomics Cat# 1000261). The labeled cells were washed with 500μL of FACS buffer and resuspended in 500μL of FACS buffer, strained through 40μm strainer prior to sorting on BD FACSAria Fusion sorter. Cells were resuspended to achieve approximately 1,000 cells/µL. 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' v2 Chemistry kits for GEMs generation. Following the Chromium Single Cell 3′ Reagents Kits version 2 user guide (CG00052 Rev B), cells were loaded to achieve approximately 10,000 cells for capture. Libraries were sequenced on the or NovaSeq 6000 platform to achieve an average of read depth of 50,000 mean reads per cell.