Truseq stranded mrna lt sample prep kit set a

RNA quality was assessed using an Agilent BioAnalyzer (Agilent). cDNA library was prepared by Illumina TruSeq Stranded mRNA LT Sample Prep Kit – Set A (Illumina, RS-122-2101) according to the manufacturer’s instructions. The library was pooled and sequenced using Illumina HiSeq 4000 with single-end 50 bp read length at The Sequencing and Genomic Technologies Shared Resource of Duke Cancer Institute. The differential analysis was performed using DESeq2 [39 (link)]. For cDNA microarray, cDNA was amplified with Ambion MessageAmp Premier RNA Amplification kit (ThermoFisher Scientific, AM1792). The gene expression signatures were interrogated with Affymetrix U133A gene chips and normalized by the RMA (Robust Multi-Array) algorithm. cDNA synthesis and microarray interrogation were performed by the Duke Microarray Core.

Using a Qiagen RNeasy Mini Kit (Cat. No. 74104), we extracted RNA from each sample according to the manufacturer's instructions. We examined RNA integrity and quantity using a NanoDrop 1000 and a 2200 TapeStation system using RNA ScreenTape (Agilent, Cat. No. 5067‐5576). A total of 5 μg RNA with an RNA integrity number (RIN) > 7 was used for RNA‐Seq library construction. Additionally, a complementary deoxyribonucleic acid (cDNA) library was prepared with the TruSeq Stranded mRNA LT Sample Prep Kit—Set A (Illumina, Cat. No. RS‐122‐2101) and amplified using polymerase chain reaction (PCR), specifically the Illumina HiSeq 2500 Sequencing System with a HiSeq SBS sequencing kit (Illumina Inc.). Resulting paired‐end reads were 100 base pairs in length and sequenced on one lane from each end for 101 cycles. We generated and demultiplexed FASTQ data files with the bcl2fastq v1.8.4 Illumina Conversion Software (Illumina Inc.). We used RNA‐Seq to analyze liver and retropharyngeal lymph node tissue samples from CWD‐positive and CWD‐ND deer. Low integrity RNA samples (i.e., RIN < 7.0) were excluded from RNA‐Seq analyses.

The total RNA was extracted using RNeasy Plant Mini Kit (Qiagen, Valencia, CA) and on-column DNase digestion was performed according to manufacturer's protocol. The concentration and quality of RNA were checked by a Nanodrop 1000 (Grand Island, NY) and by running the product on an agarose gel. The RNA-Seq libraries were prepared using a TruSeq Stranded mRNA LT Sample Prep Kit—Set A (Catalog #: RS-122-2101, Illumina, USA) according to the manufacturer's instructions. Three biological replicates were used for RNA-Seq and real-time PCR analysis.