GSM8436478: LN_CD8 VDJ; Mus musculus; RNA-Seq
Six homozygous P301S mice (28-30 weeks) were harvested and perfused with chilled normal saline. Spinal cords from each animal were dissected and two spinal cords were combined per tube for processing. All three mediastinal lymph nodes were also collected per mouse and combined into one tube for processing. Tissues for both experiments were then processed using the Miltenyi Tumor Dissociation Kit (#130-096-730, Miltenyi Biotec) and the gentleMACS™ Octo Dissociator with Heaters on the soft/medium program (37C_m_TDK_1). Following  spinal cord dissociation, myelin was removed using Myelin Removal Beads II (#130-096-731, Mitenyi Biotec) according to the manufacturer's protocol. The spinal cord and lymph node derived cell suspensions were stained the following antibodies obtained from Biolegend (BL) or BD Biosciences (BD): CD11b BV570 (1:200, #101233, BL), CD45 BUV395 (1:200, #564279, BD), CD4 FITC (1:200, #100510, BL), CD44 BUV737 (1:200, #612799, BD), CD8 BUV805 (1:200, #612898, BD) as well as LiveDead fixable Blue Cell Staining kit (1:500 in PBS, #L34962, ThermoFisher).  CD8+ and CD4+ T cells were then sorted using a BD S6 Symphony Cell Sorter into X-vivo 15 Serum-free Hematopoietic Cell Medium (Lonza cat#02-053Q). For both experiments, single cell suspension quality, number and viability were assessed with the dual fluorescence cell counter Luna-FX7 (Logos Bio). Gel Bead-in-Emulsions were prepared by loading up to 10,000 cells per sample onto the 10X Genomics and Chromium Next GEM Chip K (cat#1000287) and run using the Chromium Controller (10x Genomics version 5.0). Barcoded single cell cDNAs and DNA libraries were generated with the 10X Genomics Chromium Next GEM Single Cell 5' Kit v2 (cat#1000265) and the Chromium Single Cell Mouse TCR Amplification Kit (cat#1000254) following the manufacturer's standard protocol.