KAPA Stranded mRNA-Seq Kit

Protocol tips

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	The KAPA Stranded mRNA-Seq Kit is designed for both manual and automated NGS library construction from 100 ng – 4 µg of intact, total RNA.

Protocol tips
The KAPA Stranded mRNA-Seq Kit is designed for both manual and automated NGS library construction from 100 ng – 4 µg of intact, total RNA.

Publication protocol

Libraries for mRNA sequencing were prepared using KAPA Stranded mRNAseq Kit (Kapa Biosystems) following manufacturer’s instructions (Version 2.14). Briefly, total RNA (1.5 μg) was depleted of ribosomal RNA using supplied mRNA capture magnetic beads. Eluted mRNA was fragmented to a size of 300–400bp by incubating at 85°C for 6 minutes followed by 1st strand and 2nd strand cDNA synthesis. From here on, AMPure XP beads (Beckman Coulter) were used for subsequent cleanup wherever required. Double-strand cDNA was marked by A-tailing followed by ligation with Illumina barcoded adapters (5 μL). The libraries of six different indexes were pooled for multiplexing and analyzed by Bioanalyzer. A final 10 nM of pooled libraries were sequenced for 30 to 60 million 50-bp paired-end reads on Illumina Hi-seq 2500 platform (Illumina). TopHat was used for mapping sequence reads to the human genome (hg19). BedTools and bedGraphToBigWig generated stranded binary wiggle files after in-house scripts split reads mapped to each strand. FeatureCounts (24) computed the number of reads mapped to each gene and transcript, followed by DESeq for differential expression analysis. In-house scripts and R Bioconductor 2.15.1 were used to compare TCGA data and generate heatmaps and Venn diagrams.

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Manufacturer protocol

Download the product protocol from Roche Lifesciences for KAPA Stranded mRNA-Seq Kit below.

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