title: Illumina HiSeq 2500 sequencing; Down-regulatory responses to heat stress and lipopolysaccharide in the chicken thymic transcriptome
design: Down-regulatory responses to heat stress and lipopolysaccharide in the chicken thymic transcriptome
library_construction_protocol: In each replicate, birds were transferred at 17 days of age into pens within 4 temperature controlled chambers (2 for heat stress and 2 for thermoneutral).  Both chicken lines (broiler and Fayoumi) were included in all 4 chambers.  Birds were allowed to acclimate for 5 days at 25°C before the start of the treatment protocol. Birds were euthanized by intravenous injection of sodium pentobarbital (Fort Dodge Animal Health, Fort Dodge, IA). Tissue samples from the thymus were collected into RNAlater (Ambion, Inc., Austin, TX), perfused for 24 hours at room temperature, and then stored at -80°C to preserve the RNA. Birds from both lines (broiler and Fayoumi) were assigned to four treatments: Thermoneutral + Saline, Thermoneutral + LPS, Heat + Saline, and Heat + LPS. At 22 days of age, birds in the Heat groups were exposed to 35°C for 7 hours. Birds in the Thermoneutral groups remained at 25°C. At the midpoint of heat stress (3.5 hours), birds in the LPS groups were given a subcutaneous injection of lipopolysaccharide (100 µg of LPS/kg of body weight based on average weight for each line at day 21). Birds in the Saline groups were injected with an equivalent volume of phosphate buffered saline. Total RNA was isolated from each thymus sample using the RNAqueous Total RNA Isolation kit (Ambion, Inc., Austin, TX). Resulting RNA samples were DNase treated using the DNA-free DNA Removal kit (Ambion, Inc.). RNA quality was determined (all had RNA Integrity Score > 8.3) using the Eukaryote Total RNA Nano kit on the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Individual cDNA libraries were constructed from 0.5 µg of total RNA/sample using the Illumina TruSeq RNA Library Preparation kit version 2 (Illumina, Inc., San Diego, CA). Quality of cDNA libraries was confirmed using the DNA 1000 kit on the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
attributes.tag: Experimental Factor: compound
attributes.value: none
attributes.tag: Experimental Factor: breed
attributes.value: Fayoumi
attributes.tag: Experimental Factor: environmental stress
attributes.value: warm/hot temperature regimen