GSM9309087: Ctrl1; Mus musculus; RNA-Seq
Following euthanasia, tibiae were quickly cleaned of connective tissue and flash frozen. Prior to RNA extraction, the ends of tibiae were cut and marrow was flushed with 1X PBS (no Ca/Mg) quickly, and cryo-pulverized. Pulverized cortical bone of tibiae was transferred into Trizol reagent. RNA was then extracted following the Trizol protocol. 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 using either dUTP for directional library or dTTP for non-directional library. For the non-directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. For the directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification.