title: GSM4365519: EAE CD11b+ ROS- replicate 2; Mus musculus; RNA-Seq
library_construction_protocol: Mice were perfused with ice-cold phosphate-buffered saline (PBS) and spinal cords were flushed out from the spinal column with PBS. Tissues were incubated in HBSS with 0.2% collagenase, type 3 (Worthington) gently shaking for 30 min at 37 °C, and then mechanically dissociated and passed through a 40-um strainer (Falcon). Cells were collected in complete RPMI-1640 medium (without phenol red supplemented with 25 mM HEPES pH 7.4, 1% Penicillin/Streptavidin and 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific)) and myelin was depleted following the myelin removal beads II manufactures guidelines (Miltenyi Biotec). Myelin-depleted cell suspensions were treated for 5 min at 4 °C with Fc-block in BSA staining buffer (BD Biosciences) and then incubated for 30 min at 4 °C with CD11b APC-Cy7 (M1/70) antibodies. Live cells isolated from spinal cord were stained for intracellular ROS in vitro using membrane permeable, fluorescent reagent 2′,7′–dichlorofluorescein diacetate (DCFDA; Abcam). Cells were incubated with 10 uM DCFDA in PBS supplemented with 2% FBS without EDTA at 4 °C for 30 min prior to cell sorting. Cells were incubated for 5 min at 4°C with 1 uM sytox blue live/dead stain and then sorted using FACSAria II. Sorted cells were kept on ice until all samples were collected and then resuspended in cold PBS supplemented with 2% FBS at 233 cells/uL and immediately processed for single cell RNA-sequencing. Libraries were prepared following manufactures instructions for the Chromium Single Cell 3′ v2 Reagent Kit (10X Genomics)