GSM5469918: APP-PS1_brain, C27; Mus musculus; RNA-Seq
Brain: Brain samples were split in two hemispheres and both hemispheres per mouse were prepared. In short, each brain hemisphere (including brainstem and cerebellum) was mechanically chopped with a razor blade and homogenized in 2 mL ice cold HBSS with 15 mM HEPES (all from Thermofisher), 0.5% glucose (Sigma) and RNAsin (1:250, Promega) using a glass homogenizer. Cells were passed through a 100 µm cell strainer and rinsed two times with 2 mL ice cold HBSS (with 15 mM HEPES and 0.5% glucose). Cell suspensions were centrifuged at 340 g for 7 min. at 4°C. Myelin was removed by resuspending the cell pellet in 30% percoll (Sigma) solution and centrifuged at 950 g for 20 min. at 4°C. Supernatant was carefully removed and pellets containing cells of interest were diluted in HBSS followed by centrifugation at 300 g for 10 min at 4°C. Pellets were resuspended in PBS containing fixable viability dye eFlour780 (1:2000, eBioscience #65-0865) for 3-10 min. at RT and transferred to round-bottom polystyrene tubes (Corning). After centrifugation for 5 min. at 300 g, cell pellets were dissolved in FACS buffer (PBS with 2% BSA and 2 mM EDTA) containing Rat Anti-Mouse CD16/CD32 Fc-Block (1:100, BD Biosciences, #553141) for 5 min. at RT. Primary fluorescent-labeled antibodies were added and antibody incubation was performed for 45 min. at 4°C in the dark. Samples were washed in FACS buffer two times and centrifuged at 400 g for 5 min. Finally, cell pellets were resuspended in 800 µL FACS buffer with RNAsin (1:250, Promega #N2115) and filtered with a 30 µm cell strainer.  Blood:  Blood samples were incubated for 7-10 minutes with 1x RBC lysis buffer (eBiosciences, #00-4300) for lysis of red blood cells. Reaction was stopped with FACS buffer and samples were centrifuged for 5 min. at 500 g. Supernatant was removed and cell pellets resuspended in PBS containing fixable viability dye eFlour780 (1:2000, eBioscience #65-0865) for 3-10 min at RT. Samples were centrifuged for 5 min. at 500 g and supernatant was removed. Afterwards cell pellets were dissolved in FACS buffer containing Rat Anti-Mouse CD16/CD32 Fc-Block (1:100, BD Biosciences, #553141) for 5 min. Primary fluorescent-labeled antibodies were directly added to the samples and samples were incubated for 30 minutes at RT in the dark. Samples were washed with FACS buffer and filtered through a 30 µm cell strainer. After final centrifugation for 5 minutes at 500 g, cell pellets were dissolved in 800 µL FACS buffer with RNAsin (1:250, Promega #N2115). Samples were placed on 4 °C until they were analysed by flow cytometry. Following primary antibodies were used: CD3-PE (17A2) (1:100, BD Biosciences #555275), CD8a FITC (1:100, eBioscience #11-0081). Single stains and isotype controls were performed with PBMCs for compensation and verification of the signals. The following isotype control antibodies were used: PE IgG2b kappa (1:100, BDBiosciences #553989), FITC IgG2a kappa (1:100, eBioscience 11-4321-80). Single, living CD3 and CD8 double positive cells were gated (see, Figure 1D) and sorted from brain and blood samples via flow cytometry-based cell sorting (FACS sort) using the BD FACS Aria cell sorter (BD Biosciences) with BD FACS Diva software (8.0.1, BD Biosciences). In general about 104 CD3+/CD8+ T-cells were sorted from total brains or blood samples directly in RNA later (Sigma) and stored at -80°C. Sort purity was analyzed with sorted samples for every experiment day using LSR Fortessa flow cytometer (BD Biosciences). Sorted samples had a purity of about 97-100 %. RNeasy Micro Kit (Qiagen) was used to isolate the RNA from the sorted CD8+ T-cells. Isolation procedure was performed according to the manufacturer's instructions using an elution volume of 14 µl. The elution step was carried out twice to increase the yield. The mRNAseq was performed by Qiagen using QIAseq UPX 3' Transcriptome analysis according to the companies protocols. 3' Transcriptome analysis