GSM8172116: Fcgr2b_KO-PD-1; Mus musculus; RNA-Seq
Brains were harvested and single cells were obtained using a Tumor Dissociation Kit (Miltenyi Biotec) following the manufacturer's instructions. Brain cells were separated from debris using Percoll (GE Healthcare Life Sciences) density gradient centrifugation and were treated with ammonium-chloride-potassium lysis buffer to eliminate red blood cells. Cell suspensions were pooled from five brains/group, and blocked with anti-CD16/CD32 (clone 2.4G2). Cells were stained with PE anti-mouse CD45.2 (eBioscience) antibody and 7-AAD (BioLegend) at 4°C for 30 min. After washing with DPBS containing 1% FBS, CD45.2+7-AAD- live lung immune cells were isolated using a BD FACSAria II (BD Biosciences). Sorted cells were counted and resuspended to perform library preparation using Chromium Next GEM Single Cell 5' v2 (Dual Index) kits (10x Genomics) and Chromium Next GEM Single Cell VDJ library v2 kits (10x Genomics) according to the manufacturer's protocol. Libraries were sequenced using a HiSeq X ten (Illumina) platform for 10,000 cells.