GSM8989148: D1123; Mus musculus; RNA-Seq
Between 11am and 1pm EST, the aorta was perfused with 5mL room temperature 1x PBS through the right ventricle of the heart. The descending thoracic aorta (proximal to the left subclavian down to the diaphragm) was collected and the adventitia was stripped from the aortas using forceps. The aortas were then placed into a screw cap tube containing 700uL TRIzol (ThermoFisher Scientific 15596018) plus 4-6 3mm zirconium (X) beads and flash frozen in liquid nitrogen. The tubes were then stored at -80°C until further processing. Within a treatment group (N = 3), aortas were randomized to determine processing order. Aortas were processed in batches containing 1 aorta per treatment group (total aortas per batch N = 10). Then, RNA was extracted using the PureLink RNA Mini Kit (Invitrogen 12-183-018A) according to the manufacturer's instructions with the following modifications. For the “Homogenization” section of the protocol: The aortas were thawed on wet ice and homogenized using the FastPrep-24 5G bead beating grinder and lysis system using the following settings: 5 cycles of 4.5m/sec for 30sec with 300sec pause time using Lysing Matrix A at 1mg. Then 200uL chloroform (Fisher Scientific 02-002-590) was added and shaken vigorously for 15sec then incubated at room temperature for 3min. Samples were then spun at 4°C for 15min at 12,000xg. During the “Binding, Washing, and Elution of RNA” section of the protocol: 350uL of ice cold 70% ethanol was used in step 1 and only 350uL of Wash Buffer I was used in step 6. After step 7, 80uL of DNase I (70uL buffer RDD + 10uL DNase I) from RNase-Free DNase set (Qiagen 79254) was added to the column and incubated at room temperature for 15min. Step 6 was then repeated. Samples were eluted into 100uL of nuclease free water and spun down once in step 14, then they were stored at -80°C. Samples were submitted on wet ice to the Sidney Kimmel Comprehensive Cancer Center Experimental and Computational Genomics Core at Johns Hopkins for QC using the Agilent 2100 BioAnalyzer and cDNA synthesis. Briefly, the TruSeq Stranded Total RNA Library Kit was used according to manufacturer's instructions to generate barcoded samples. The library was sequenced on the NovaSeq6000 S4 (150bp PE), targeting 50K reads/sample.