GSM8797472: Rag2KO-hCSF1KI-AppKI 4; Homo sapiens; RNA-Seq
At 6 months of age the human microglia were extracted from the mouse brain using the Neural Dissociation Kit (P; Miltenyi, 130-092-628) following manufacturer's instructions. After myelin removal using 30% Percoll, the cell pellets were washed (DPBS with 2% FBS & 2mM EDTA) and blocked with FcR blocking solution (1/10; Milteny, 130-092-575) and subsequently stained with the viability dye eFluor780 (1/1000; Thermo Fisher 65-0865-14) and the antibodies anti-CD11b (1/50; Miltenyi 130-113-806), anti-human CD45 (1/50; BD Bioscience 555485) and anti-mouse CD45 (1/200; BD Bioscience 563890). Human microglia (defined as CD11b positive and human CD45 positive)  were sorted using the Miltenyi MACS Quant Tyto cell sorter, pelleted and stored at -80C until further processing. RNA extraction was performed using the Qiagen RNeasy Mini kit (Qiagen 74104) according to the manufacturer's protocol. Extracted RNA was eluted in 45ul of RNAse-free water and stored at -80C. RNA concentration, purity and integrity were determined using the Nanodrop ND-8000 and Bioanalyzer 2100 and full length cDNA was created using 1ng of total RNA as input for the SMART-Seq HT PLUS protocol tusing a 1-step RT-PCR (Takara Bio; version 011921). Next, 4ng of purified double-stranded cDNA was enzymatically fragmented and stem-loop adapters were ligated. Next, libraries were amplified and indexed, generating Illumina-compatible libraries with unique dual identifiers (UDIs) following manufacturer's protocol. Each sample sequence library was pooled equimolar and sequenced on the AVITI 2x75 Cloudbreak High, single read (101-8-8-0).