Total RNA was extracted as described in the RT-qPCR protocol section. Banks for deep-sequencing were prepared from 800 ng total RNA using Illumina's TruSeq Stranded mRNA LT Kit according to manufacturer's protocol. Samples were sequenced paired-end at 50-bp read length on an Illumina HiSeq-2000. As described in supplementary methods , the reads were trimmed using Trimmomatic, aligned onto the hg19 reference genome using TopHat2, filtered using SamTools, then the FPKM calculated using Cufflinks (21 –24 (link)).
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RNA-Seq Library Preparation and Sequencing
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Corresponding Organization : Université de Sherbrooke
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Variable analysis
independent variables
- Total RNA extraction method
- Library preparation using Illumina's TruSeq Stranded mRNA LT Kit
- Sequencing on Illumina HiSeq-2000 platform
- Read trimming using Trimmomatic
- Alignment to hg19 reference genome using TopHat2
- Filtering using SamTools
- FPKM calculation using Cufflinks
- Transcriptome profiling
- Not explicitly mentioned
- No positive or negative controls specified
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