title: GSM2463008: WT2 r2; Mus musculus; RNA-Seq
library_construction_protocol: Mice from WT, C5aR1KO, Arctic and Arctic/C5aR1KO genotypes were perfused with PBS at 2, 5, 7 and 10 mo, and the brains (without olfactory bulbs, cerebellum, or midbrain) collected for microglia isolation. Brains were treated with Dispase II (Roche) at 37˚C for 1 hr and passed through a 70 µM nylon cell mesh strainer (BD Biosciences) to obtain a single cell suspension. The cell suspension was washed in Hanks’ balanced salt solution (HBSS; ThermoFisher) and spun at 1000 x g for 10 min at 4˚C. The cell pellet was resuspended in 70% Percoll (Sigma; diluted in HBSS), and 35% Percoll (diluted in HBSS) was carefully layered over to create a discontinuous Percoll gradient.  After centrifugation at 800 x g for 45 min at 4˚C, the cells at the interphase were quickly collected, washed with HBSS + 2% FBS and passed through a 40 µM nylon mesh cell strainer. Using a FACS Aria II, the viable cell population from a propidium iodide stained control was gated based on forward and side scatter properties. Using these forward and side scatter parameters, the GFP+RFP+ and the GFP+RFP- cells were sorted and collected. Total RNA from and GFP+RFP- cells was extracted for library construction. Total RNA from and GFP+RFP- cells was extracted using the Illustra RNAspin mini isolation kit (GE Healthcare). For qRT-PCR, RNA was converted to cDNA using Superscript III reverse transcriptase (Life Technologies) following manufacturer’s instructions. RNA was quantified using the NanoDrop ND-1000 spectrophotometer and the quality checked using the Agilent Bioanalyzer 2100. The SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian (Clonetech Laboratories) was used to generate Illumina-compatible RNA-seq libraries. Briefly, total RNA was converted into cDNA and then adapters with barcodes for Illumina sequencing were added by PCR.  The PCR products were then purified and the ribosomal cDNA depleted with sequence specific probes and RNaseH. The barcoded cDNA libraries were sequenced on the Illumina HiSeq 2500 platform to yield 100-bp paired-end reads.
attributes.tag: GEO Accession
attributes.value: GSM2463008