GSM8515726: temporo-occipital cortex, A+/Pki/ki, female, mouse 222; Mus musculus; RNA-Seq
Mouse temporo-occipital cortex homogenate was homogenized in TRI Reagent (#T9424, Sigma-Aldrich) after which chloroform was added (one fifth of the volume of TRI Reagent) and the tubes were shaken vigorously. Mixtures were incubated at room temperature for 5 min and the phase separation was performed by centrifugation at 12000× g for 20 min at +4 oC. RNA was precipitated from the aqueous phase with 2-propanol by mixing and incubating at room temperature for 30 min. Samples were centrifuged at 12000× g for 25 min at +4 oC and the pellets were washed twice with 75 % ethanol. RNA pellets were air-dried and dissolved in RNAse-free H2O. Isolated mouse CD11b+ microglia were resuspended in DPBS and immediately mixed with lysis buffer of the High Pure RNA Isolation kit (#11828665001, Roche). RNA was extracted following the kit instructions and sent for sequencing to Novogene Europe (below). Library preparation and RNA sequencing was conducted by Novogene (Cambridge, UK) and executed with an Illumina high-throughput sequencing platform. mRNA enrichment was performed with oligo(dT) bead pulldown, from where pulldown material was subjected to fragmentation, followed by reverse transcription, second strand synthesis, A-tailing, and sequencing adaptor ligation. The final amplified and size selected library comprised of 250–300 bp insert cDNA and paired-end 150 bp. Sequencing yielded reads for 20.2–31.34 and 29.4–49.9 million library fragments per CD11b+ mouse microglia, and mouse cortex, respectively.