GSM5463953: Saline replicate 3; Mus musculus; RNA-Seq
Brains from PBS-perfused mice were collected and microglial cells (Cd11b+, CD45low, and CD206-) were sorted on the FACS ARIA (BD Immunocytometry Systems). Total RNA was extracted using the RNeasy Plus Micro Kit (Qiagen) according to the manufacturer's instructions. Ion Torrent sequencing libraries were prepared according to AmpliSeq™ Transcriptome Mouse Gene Expression Kit protocol following manufacturer's instructions. Briefly, 1 ng of total RNA was reverse transcribed, the resulting cDNA was amplified for 16 cycles by adding PCR Master Mix and the AmpliSeq Mouse transcriptome gene expression primer pool. Amplicons were digested with the proprietary FuPa enzyme, then barcoded adapters were ligated onto the target amplicons. The library amplicons were bound to magnetic beads, and residual reaction components were washed off. Libraries were amplified, re-purified and individually quantified using Agilent TapeStation High Sensitivity tape. Individual libraries were diluted to a 50 pM concentration and pooled equally. Emulsion PCR, templating and 550 chip loading was performed with an Ion Chef Instrument (Thermo-Fisher). Sequencing was performed on an Ion S5XL™ sequencer (Thermo-Fisher).