GSM6622851: Cav1 knockout (KO) week 70 aging brain rep1; Mus musculus; RNA-Seq
Total RNA was isolated from day 15 fetal brain of WT and KO mice, in three biological replicates, using an AllPrep DNA/RNA Mini Kit (Qiagen, Cat No./ID: 80204) following the manufacturer's instruction. Sample was homogenized with 500 ul -1 ml RLT buffer (Qiagen, Cat No./ID: 79216) supplemented with 5μl of 2-mercaptoethanol. The homogenate was transferred to a fresh tube and centrifuged for 1 minute at ≥ 8000 x g. From the supernatant, 750μl was transferred to fresh tube and mixed with 1 volume 70% ethanol to precipitate RNA. RNA was eluted in 30μl nuclease-free water twice to a total volume of 60μl. Concentration of RNA was determined using Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific). RNA Integrating Number (RIN) was determined (using Agilent2100). Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification.