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

Total RNA was extracted from fresh cells or frozen tumors using TRIzol® Reagent (Invitrogen), or AllPrep DNA/RNA Mini Kit (Qiagen) or NucleoSpin RNA kit (Macherey-Nagel; for the SKN-SH cell line treated with chemotherapy). All samples were subjected to quality control on a Bioanalyzer instrument and only RNA with RIN (RNA Integrity Number) > 6 were used for sequencing. RNA sequencing libraries were prepared from 1 µg of total RNA using the Illumina TruSeq Stranded mRNA Library preparation kit which allows performing a strand-specific sequencing. A first step of polyA selection using magnetic beads is done to focus sequencing on polyadenylated transcripts. After fragmentation, cDNA synthesis was performed and resulting fragments were used for dA-tailing and then ligated to the TruSeq indexed adapters. PCR amplification is finally achieved to create the final cDNA library. After qPCR quantification, sequencing was carried out using 2 x 50 cycles (paired-end reads 50 nts) for all samples (except SH-EP, 2 x 100; Pair1-Relapse and Pair3-Relapse, 2 x 75; Pair2-Relapse, 2 x 150). Sequencing was performed with the Illumina HiSeq2500 instrument (high output mode) except for cases Pair1-Relapse and Pair2-Relapse analyzed with the NextSeq500 instrument and Pair3-Relapse3
analyzed on a HiSeq4000 instrument. Reads were aligned to the human reference genome hg19/GRCh37 using TopHat2 v2.0.645 with the following parameters: global alignment, no mismatch in the 22 bp seed, up to three mismatches in the read, library type fr-firststrand. Gene expression values (FPKM=fragments per kilobase per million reads) were computed by Cufflinks v2.2.146 and further normalization between samples was done using quantile normalization (R/Bioconductor package limma)

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Manufacturer protocol

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