Dna rna miniprep kit

Mice were anesthetized with Avertin (2.5%) until unresponsive to stimuli, then perfused with Hank’s Balanced Salt Solution (HBSS^−/−) with 1 mM Hepes. The brain was extracted, hemisected, and the forebrain was placed in Accutase (Millipore, SRC005) to be mechanically processed and enzymatically dissociated for 30 min at 4 °C while shaking. Tissue was mechanically dissociated further with a serological pipette, then a micropipette, and strained through a 250 μm filter. The single cell suspension was resuspended in 100% Fetal Bovine Serum then in a 30% Percoll solution overlayed with FACS Media (10% FBS, 1 mM Hepes in HBSS^−/−). The slurry was centrifuged at 800×g for 15 min with slow acceleration and break. The Percoll layer was then aspirated away from the pellet and cells were washed once in FACS Media. Cells were stained for CD45 expression with PE-Cy7-Rat anti-mouse CD45 antibody (BD Pharmigen; Clone 30-F11, catalog number: 552848) and with DAPI (Sigma). DAPI negative, CX3CR1-YFP⁺ and Pe-Cy7-CD45^int (microglia) cells were isolated and collected by FACS. Microglia were then pelleted at 2500 rpm at 4 °C for 5 min, then lysed in buffer from the DNA/RNA Mini-prep kit (Zymo;11-385). Lysed samples were frozen at − 80 °C until DNA/RNA was extracted.

RNA was isolated from liver tissues stored at −80 °C by first homogenizing using a Dounce homogenizer in DNA/RNA shield (provided by Zymo ZR-Duet kit), incubated with protease K (provided by kit) for 30 min at 55 °C, then column extracted using the Zymo ZR-Duet DNA/RNA MiniPrep kit following the manufacturer’s instructions for silicon column-based RNA and gDNA extraction. Genomic DNA was extracted from liver samples using Qiagen DNase kit according to the manufacturer’s instruction.