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

Three biological replicates were prepared for RNA-seq with the TruSeq stranded mRNA library preparation kit (Illumina) and sequenced on the Illumina HiSeq 2000 platform. FastQ sequences were mapped to the mouse mm9 genome by StrongARM, developed for the Pediatric Cancer Genome Project (Downing et al. 2012). Mapped reads were counted with HTSeq (Anders et al. 2015), and gene-level FPKM (fragments per kilobase per million mapped fragments) values were computed. All sample data were collated into a matrix using R (3.0.1) and log start-transformed [log2(FPKM + 1] in STATA/MP 11.2. Genes were statistically tested by class (t-test, unequal variance t-test per gene). Bonferroni correction and false discovery rates (FDRs) were determined to allow both strict and modest multiple comparison filtering (Partek Genomics suite 6.6). Six-hundred-fifty-five genes were selected that had an absolute value log ratio of at least 1 in at least one comparison and passed FDR at 5% (vs. respective controls). We then filtered for genes that showed concordant regulation with MLX-WT, MLX-DN, and MLX shRNA; i.e., genes that change in the same directions in MLX-DN and MLX shRNA samples and do not have the same directional change in the MLX-WT samples. The 80 genes concordantly regulated by MLX were identified because they satisfied a minimum of two out of three of the following requirements: a significant change of at least 1.5-fold compared with control for (1) MLX-WT, (2) MLX-DN, and (3) MLX shRNA; that is, the 80 selected genes were either regulated consistently by both MLX-DN and MLX shRNA or in an opposite fashion by MLX-WT and MLX-DN and/or MLX shRNA. These were analyzed for enriched gene sets by using the SwissProt PIR (SP_PIR) and DAVID. RNA-seq results are available at the Gene Expression Omnibus with accession number GSE70028.

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