Illumina HiSeq 4000 sequencing; Identification of exercise-related genes in mice exposed to cigarette smoke
Identification of exercise-related genes in mice exposed to cigarette smoke
Ten-week-old A/JOlaHsd female mice (Harlan Laboratories, UK) were used in this study, as approved by the Norwegian Animal Research Authority and following ACSM's animal care standards. Ten week old A/JOlaHSD female mice (Harlan Laboratories, Loughborough, UK) were randomly divided into three groups and exposed to room air (n=10), CS (n=10) or CS plus HIIT (n=11). CS exposure (Research Cigarettes 3R4F, University of Kentucky, Lexington, KY, USA) was performed for six hours per day, five days per week for 20 weeks in an exposure chamber (TE-10, Teague Enterprises, Woodland, CA, USA). After CS exposure was completed, HIIT was performed five times per week for six weeks on a treadmill with 25 incline and gradually increasing speed as the training programme progressed. At sacrifice, the heart (left ventricle (LV) and right ventricle (RV)) and left lung were dissected, and immediately frozen in liquid nitrogen and stored at -80 for subsequent profiling.  Total RNA was extracted from 91 (RV (n=29), LV (n=31) and lung (n=31)) mouse tissue samples, using RNeasy® Fibrous Tissue Mini Kit (Qiagen Sciences Inc., Germantown, MD, USA) and RNeasy Mini Kit (Qiagen) respectively, according to manufacturer´s instruction. RNA quantity, quality/integrity and purity were evaluated using Qubit (Thermo Fisher Scientific, Waltham, MA, USA), Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and NanoDrop (Thermo Fisher). The libraries were quantified (KAPA Library Quantification Kit (Illumina/ABI Prism)), normalized and pooled. Quantitated libraries were further diluted to 2,4 nM and subjected to clustering by a cBot Cluster Generation System on one HiSeq4000 flowcells (Illumina Inc., San Diego, CA, USA), according to manufacturer´s instructions. Finally, single-end read sequencing was performed for 75 cycles on an Illumina HiSeq4000 instrument, by the manufacturer's instructions (Illumina).