GSM8169781: bone_Y-Ctrl1-CR-11_tech_rep1; Mus musculus; RNA-Seq
Tibia bones were isolated by completely removing adherent muscle and connective tissue, and were immediately frozen in liquid nitrogen and stored at -80°C until further processing. RNA was extracted from whole-tissue, ground to a powder while frozen in liquid nitrogen, using the phenol/chloroform method. Specifically, an aliquot of approximately 50 mg of tissue powder was vortexed for one minute with 200 µl of Trizol (Thermo Fisher Scientific). After centrifugation at 12,000g for 10 minutes at 4°C, supernatant was transferred to a new tube and 40µl of chloroform were added. Samples were centrifuged for 15 minutes at 12,000g and 4°C, followed by RNA precipitation from the collected upper clear phase with 250 μl of isopropanol. RNA was purified by washing in 500 μl of 75% ethanol, dried and solubilized in 30 μl DEPC-treated, i.e. RNAse-free, water. DNase digest of purified RNA was performed using the Invitrogen Turbo DNA-free kit (Thermo Fisher Scientific). An Agilent 2100 BioAnalyzer (Agilent RNA 6000 Nano Kit) was used to check for total RNA quality of the samples. mRNA was prepared for sequencing using the TruSeq stranded mRNA sample preparation kit (Illumina), with a selected insert size of 120–210 bp, and sequenced on an Illumina HiSeq 4000 instrument.