title: GSM8806180: WT2-STAT; Mus musculus; RNA-Seq
library_construction_protocol: Twelve-month-old WT and APP mice were killed and their PFC rapidly collected in Hank's Balanced Salt Solution without Ca2+ and Mg2+ (HBSS; Sigma). The PFC of three mice were pooled, resulting in N=5 independent PFC samples from APP mice. Cells were mechanically and enzymatically dissociated with fire-polished Pasteur pipettes and the neural tissue dissociation kit with papain (Miltenyi Biotec), following manufacturer's instructions. Myelin removal beads II (Miltenyi Biotec) were used to deplete myelin from cell suspensions using MS columns. Cells were centrifuged at 300 g for 5 min at 4°C, resuspended in 400 µl HBSS with Ca2+ and Mg2+ and sorted on a BD Influx cell sorter. GFP was detected at 530/40 nm (488 nm excitation), CFP at 450/50 nm (408 nm excitation). The complete protocol is described in Ceyzériat et al. 2018 (PMID 30322407). Sorted cells were placed 5 min at RT and chloroform was added to TRIzol for 3 min. Samples were centrifuged at 12,000 g for 15 min at RT. Aqueous phase was collected and 1 volume of 70% ethanol was added. Samples were transferred onto an RNeasyMin Elute spin column and RNA was purified according to manufacturer's instructions, with on-column DNAse treatment (RNeasy micro kit, Qiagen, Hilden, Germany). RNA was eluted with 14 µl of RNAse-free deionized water and stored at - 80 °C until transcriptomic analysis. The lower number of astrocytes sorted per sample was not compatible with RNA quality assessment, and full length double strand cDNA libraries were amplified with 18 LD-PCR cycles.
identifiers.namespace: GEO
identifiers.id: GSM8806180_r1