title: GSM6198282: 2m_SPF_biol rep 2; Mus musculus; RNA-Seq
library_construction_protocol: Young and old GF and SPF mice (young GF, 2 months, N = 2; young SPF, 2 months, N   = 2; old GF, 22 months, N = 2; old SPF, 22 months) were anaesthetized by intraperitoneal injection of three anaesthetic agents and heparin and transcardially perfused with ice-cold HBSS containing heparin. The brains were cut in sagittal midlines and the cerebral hemispheres were roughly minced by scissors, followed by digestion according to the standard protocol of Neural tissue dissociation kits (P). The brain homogenate was filtered through a 70 µm cell strainer, and cell debris was removed by centrifugation according to the standard protocol of Debris Removal Solution. The cells in HBSS with 1% BSA were then treated on ice with CD16/32 FcR-blocking reagent for 10 minutes, followed by 15 minutes of staining with fluorescence-conjugated antibodies. The following antibodies were used (all antibodies were purchased from BioLegend): Fluorescein isothiocyanate (FITC)-conjugated anti-Cd45 (cat#: 103108) and allophycocyanin (APC)-conjugated anti-Cd11b (cat#: 101212). Before sorting, cells were filtered through a 40 µm cell strainer and stained with propidium iodide (PI). PI-negative, Cd45-low, and Cd11b-positive cells were sorted into 1.5 ml Protein LoBind Tubes containing 1% BSA in HBSS and then centrifuged. After removal of the supernatant, the remaining pellet was lysed and total RNA was isolated according to the standard protocol of the RNeasy Micro Kit. Full-length cDNA was prepared using a SMART-Seq HT Kit (Takara Bio, Japan) according to the manufacturer's instructions. According to the SMARTer kit instructions, an Illumina library was then prepared using a NexteraXT DNA Library Preparation Kit (Illumina).