TruSeq Stranded mRNA

Protocol tips

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	The protocol is optimized for 0.1–1 µg of total RNA.

Protocol tips
The protocol is optimized for 0.1–1 µg of total RNA.

Publication protocol

RNA was isolated from three separate experiments for each cell line and treatment as previously reported6. The Truseq Stranded mRNA kit (Illumina) was used to prepare mRNA libraries from 1 µg total RNA. Libraries were confirmed on the Agilent 2100 Bioanalyzer and quantitated using the Illumina Library Quantification Kit, ABI Prism qPCR Mix from Kapa Biosystems and the ABI7900HT real-time PCR instrument at the University of Louisville Center for Genetics and Molecular Medicine (CGeMM) DNA Sequencing Core Facility. 76 cycle single read sequencing was performed with the 500 High-output v2 (75cycle) sequencing kit on the Illumina NextSeq. 500 instrument. The sequence reads were mapped to the human reference genome, version GRCh37.1 (hg19) using the mapping algorithm tophat11 version 2.0.2. The expression levels were quantified at loci specified by the annotation found within the ENSEMBL release 73 gene description file (Homo_sapiens.GRCh37.73.gtf) using cufflinks version 2.2.1. Contributions to the annotation file from both ribosomal RNA (rRNA) and mitochondrial RNA (mtRNA) were removed from the gtf file prior to use. Differential analyses between the specified conditions was performed using cuffdiff version 2.2.1. The raw data were uploaded in the Gene Expression Omnibus (GEO) database as GSE78011.

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Manufacturer protocol

Download the product protocol from Illumina for TruSeq Stranded mRNA below.

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