title: Illumina HiSeq 2500 paired end sequencing; Next Generation Sequencing Transcriptomic Analysis (RNA-seq) of Thoroughbred horse at rest, after exercise and after training
design: Next Generation Sequencing Transcriptomic Analysis (RNA-seq) of Thoroughbred horse at rest, after exercise and after training
library_construction_protocol: Percutaneous needle muscle biopsies (approx. 300mg) were obtained from the ventral compartment of the middle gluteal muscle from standing unsedated horses using a previously described method [53]and preserved in RNAlater (Thermo Fisher, Massachusetts, United States). Total RNA was extracted from approximately 70mg tissue, using a protocol combining TRIzol reagent (Thermo Fisher, Massachusetts, United States), DNase treatment (RNase free DNase) (Qiagen, Hilden, Germany) and RNeasy Mini-Kit (Qiagen).  The study cohort comprised a subset of Thoroughbred horses, trained for Flat racing at a single training establishment under the management of a single trainer. The UR cohort comprised of yearling Thoroughbred horses (n = 51; 23 males and 28 females; 19.5±1.5 months old) in submaximal training prior to entering sprint training. The UE cohort consisted of the same group of horses undertaking their first or second 'work day' (WD, high-intensity sprint exercise simulating a race) on an all-weather gallop. The TR cohort consisted of horses at the end of the racing season following approximately six months of sprint training. All horses in this group had achieved “race fitness” with an average of 15.1±9.1 SD WDs (range 6-43). Table 1 summarises details of sampling and physiological measurements. Percutaneous needle muscle biopsies (approx. 300mg) were obtained from the ventral compartment of the middle gluteal muscle from standing unsedated horses using a previously described method [53]and preserved in RNAlater (Thermo Fisher, Massachusetts, United States).  Total RNA was extracted from approximately 70mg tissue, using a protocol combining TRIzol reagent (Thermo Fisher, Massachusetts, United States), DNase treatment (RNase free DNase) (Qiagen, Hilden, Germany) and RNeasy Mini-Kit (Qiagen). RNA was quantified using a Nano Drop ND1000 spectrophotometer V 3.5.27 and RNA quality and purity were assessed using the 18S/28S ratio and RNA integrity number (RIN) on an Agilent Bioanalyser with the RNA 6000 Nano LabChip kit6 (Agilent, Cork, Ireland). The RNA isolated from UR, UE and TR samples had an average RIN of 8.1 (7.2-9.3), 8.2 (7-9.1) and 8.1 (5.4-8.6), respectively.  Indexed, strand-specific Illumina sequencing libraries were prepared using the TruSeq Stranded mRNA Library Preparation Kit LT (Illumina, San Diego, United States).
attributes.tag: Experimental Factor: Untrained_Exercise
attributes.value: cohort