Illumina HiSeq 4000 paired end sequencing; scRNA-seq data of mouse splenocytes
The mice were maintained under specific pathogen-free conditions at the Wellcome Trust Genome Campus Research Support Facility (Cambridge, UK). These animal facilities are approved by and registered with the UK Home Office. All procedures were in accordance with the Animals (Scientific Procedures) Act 1986. The protocols were approved by the Animal Welfare and Ethical Review Body of the Wellcome Trust Genome Campus.The spleen from a 6-month-old C57BL/6Jax mouse was removed. The splenocytes were isolated by passing the spleen through a 70 µm cell strainer (Fisher Scientific 10788201) into 30 m ice-cold 1X DPBS (Thermo Fisher 14190169) with 2 mM EDTA and 0.5% (w/v) BSA (Sigma A9418) using the plunger of a 2-ml syringe. Cells were spun down at 500 g for 7 minutes at 4 degree. Then the supernatant was removed, and the cell pellet resuspended in 5 ml 1X RBC lysis buffer (Thermo Fisher 00-4300-54). The cell suspension was vigorously vortexed for 5 seconds and left on the bench for 5 minutes to lyse the red blood cells. Then 45 ml ice-cold 1X DPBS was added, and cells were spun down at 500 g for 7 minutes at 4 degrees. The supernatant was removed, and 30 ml ice-cold 1X DPBS with 0.1% BSA was used to resuspend the cell pellet. The cell suspension was passed through a Miltenyi 30 μm Pre-Separation Filter (Miltenyi 130-041-407), and the cell number was determined using the C-chip counting chamber (VWR DHC-N01). The cells were spun down again, and the cell pellet resuspended in ice-cold 1X DPBS with 0.1% BSA to reach a concentration of 1,000,000 cells per ml. The splenocytes were then loaded on the 10x Chromium Controller, aiming to recover ~ 5000 cells (Targeted Cell Recovery 5000 cells). cDNA and a sequencing library were made according to 10x Single Cell 3' Reagent Kits v2 manual.