GSM1690103: thymus_1_dpi_negative_control_rep1; Gallus gallus; RNA-Seq
RNA samples were isolated using the Ambion MagMax-96 Kit (AM1839) (Applied Biosystems, Foster City, CA) and immediately stored at -80 . All extraction procedures were performed according to manufacturer’s recommendations. A NanoDrop ND-1000 UV-vis Spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA) was used to quantify the RNA. In addition, the quality of RNA was tested using the Agilent 2100 Bioanalyser (Agilent Technologies). The RNA Integrity Number (RIN) was greater than 9.0 for all the RNA samples applied to construct the cDNA libraries. The initial total RNA was converted into a cDNA sequencing library through the Illumina TruSeq® RNA Sample Preparation v2 Kit following manufacturer’s instructions (Protocol: #15026495, May 2012). First, 0.1 g - 4 g total RNA was purified using Poly-T oligo-attached magnetic beads to obtain the mRNA fragments. Next, the first-strand cDNA was synthesized from the mRNA fragments. Finally, second-strand cDNA synthesis, end repair, 3’ end adenylation, and adapter ligation were performed and PCR amplification was conducted. The cDNA libraries were validated and quantified using a Qubit® Quantitation Platform and an HS dsDNA kit (Invitrogen, Paisley, UK). Twenty-four cDNA libraries were generated from the twenty-four samples, in terms one cDNA library for one sample. One of the each of the six treatments was pooled into one lane to sequence. Thus, four lanes were used to sequence the twenty-four cDNA libraries through the Illumina® HiSeq 2000 at the Iowa State University (ISU) DNA facility.
GEO Accession
GSM1690103