Ambion magmax 96 kit

An Ambion MagMAX-96 Kit (AM1839) (Applied Biosystems, Foster City, CA) was used to isolate RNA from the thymus samples. The quality and quantity of RNA were assessed using the Agilent 2100 Bioanalyzer according to the manufacturer's instructions (Agilent Technologies). RNA Integrity Numbers (RIN) for all the RNA samples selected to construct the cDNA libraries were greater than 8.0. Next, an Illumina TruSeq ® RNA Sample Preparation v2 Kit was utilized to convert 0.1 to 4 μg RNA into cDNA libraries. Twenty-four cDNA libraries, which included 4 biological replicates (birds) for each treatment, were constructed. Briefly, fragment mRNA was purified using oligo-dT beads from the initial RNA and reverse transcribed into a double strand cDNA fragment. End repair, adenylation, adapter ligation, and PCR amplification were then carried out in conformance with the TruSeq® manufacturer's instructions (Protocol: #15026495, May 2012). A Qubit® Quantitation Platform and HS dsDNA kit (Invitrogen, Paisley, UK) were then used to test and quantify the cDNA libraries. Six cDNA libraries, including one for each of the 6 treatments, were sequenced in the same lane of the Illumina® HiSeq 2000 at the Iowa State University DNA facility (4 lanes for the 24 cDNA libraries) with single end 100 bp cycles.