title: Illumina HiSeq 1500 paired end sequencing; RNA-Seq of the aorta of hypercholesterolemic mice treated by either ticagrelor, clopidogrel, or control.
design: RNA-Seq of the aorta of hypercholesterolemic mice treated by either ticagrelor, clopidogrel, or control.
library_construction_protocol: After sacrifice, the blood was flushed from circulation by infusing pressurized phosphate buffered saline from the left ventricle of the animal.  The entire aortae, which included the distal portion of the ascending aortae, aortic arches, and descending aortae down to the liac bifurcations, were excised from the sacrificed animals, under dissecting microscope. The excised aortae were thoroughly cleaned under dissection microscope to be free of other tissue such as adipose, muscle, and connective tissues.  They were then stored in -80 degree until the extraction of RNA.    After sacrifice, the blood was flushed from circulation by infusing pressurized phosphate buffered saline from the left ventricle of the animal.  The entire aortae, which included the distal portion of the ascending aortae, aortic arches, and descending aortae down to the liac bifurcations, were excised from the sacrificed animals, under dissecting microscope. The excised aortae were thoroughly cleaned under dissection microscope to be free of other tissue such as adipose, muscle, and connective tissues.  They were then stored in -80 degree until the extraction of RNA.    All mice were placed on high-fat diet (HFD, 1.25% cholesterol, 40% kcal fat; D12108Ci, Research Diets, Inc., New Brunswick, NJ) at 8th week of age and maintained on the diet for 16 weeks.  HFD contained either ticagrelor, clopidogrel, or nothing (control).   The mice were fed HFD containing either ticagrelor (180mg/kg/day), clopidogrel (25 mg/kg/day), or nothing (control).  The doses of ticagrelor and clopidogrel completely inhibited the platelet aggregation by light transmission aggregometry.  Illumina software bcl2fastq v2.17.1.14 was used to convert bcl files into fastq format. Total RNA was isolated individually from each aorta using Qiagen RNA purification columns.  The quality of the RNA was examined, using the Agilent 2100 Bioanalyzer and found that all samples had RNA integrity numbers higher than 8.   RNA samples were polyA selected and paired-end sequencing libraries were constructed using TruSeq RNA Sample Prep Kit as described in the TruSeq RNA Sample Preparation V2 Guide (Illumina).
attributes.tag: Experimental Factor: compound
attributes.value: clopidogrel
attributes.tag: Experimental Factor: dose
attributes.value: 25