TruSeq RNA Library Prep Kit v2

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	0.1 - 1 ug total RNA or 10 - 400 ng previously isolated mRNA (from species with polyA tails)

Protocol tips
0.1 - 1 ug total RNA or 10 - 400 ng previously isolated mRNA (from species with polyA tails)

Publication protocol

TCR-redirected or mock-treated T cells cocultured with Huh7A2 HCVRep cells at an E/T ratio of 10:1 were collected at the indicated times and enriched with anti-CD8 MicroBeads (Miltenyi Biotech) as per the manufacturer's instructions. Enriched CD8+ T cells (purity, 98% CD3+ CD8+) at 1.5 million cells per sample were stored at −80°C. Total RNA was isolated with TRIzol reagent (Thermo Fisher Scientific, MA, USA), and RNase-free reagents were used throughout the isolation. Sample quantity and purity were assessed using the NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, MA, USA). All RNA samples had RNA integrity number (RIN) values of >9. The RNA libraries were bar coded and prepared according to the protocol Illumina TruSeq RNA Sample Prep kit v2 (catalog number RS-122-2001) from the manufacturer (Illumina, San Diego, CA, USA).
The nine RNA libraries were sequenced in paired ends on an Illumina HiSeq 2000 instrument, where each sample was put in two different lanes, for a total of 18 samples. The runs generated a total of 384 million reads with an average of 43 million paired-end reads per sample, of which 90% mapped to the human genome. The paired-end sequences were aligned to the human genome reference hg19, with TopHat2 version 0.9 using TopHat standard parameters. The numbers of aligned reads per gene were determined with HTSeq 0.6.1. Annotations from Ensembl Homo sapiens grch37.82 and Genome Browser were used to assign features to genomic positions. The R/Bioconductor package DESeq2 was used for differential gene expression on read counts generated by HTSeq 0.6.1.

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Manufacturer protocol

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