SENSE mRNA-Seq Library Prep Kit V2

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	The typical amount of input total RNA is 1 ng – 2 µg- Works with high-quality RNA from any species with polyA tails.

Protocol tips
The typical amount of input total RNA is 1 ng – 2 µg- Works with high-quality RNA from any species with polyA tails.

Publication protocol

Total RNA was isolated using Trizol reagent (Invitrogen). The RNA quality was assessed by an Agilent 2100 bioanalyzer using the RNA 6000 Nano Chip (Agilent Technologies, The Netherlands), and RNA quantification was performed using an ND-2000 Spectrophotometer (Thermo Inc., USA). For control and test RNAs, construction of the library was performed using a SENSE mRNA-Seq Library Prep Kit (Lexogen,Inc., Austria) according to the manufacturer’s instructions. Briefly, each 2 µg of total RNA was prepared and incubated with magnetic beads decorated with oligo-dT and then other RNAs except mRNA was removed with a washing solution. Library production was initiated by the random hybridization of starter/stopper heterodimers to the poly(A) RNA still bound to the magnetic beads. These starter/stopper heterodimers contain Illumina-compatible linker sequences. A single-tube reverse transcription and ligation reaction extends the
starter to the next hybridized heterodimer, where the newly synthesized cDNA insert is ligated to the stopper. Second-strand synthesis is performed to release the library from the beads, and the library is then amplified. Barcodes are introduced when the
library is amplified. High-throughput sequencing was performed as paired-end 100 sequencing using HiSeq 2000 (Illumina, Inc., The USA). mRNA-Seq reads were mapped using the TopHat software tool to obtain the alignment file. Differentially expressed genes were determined on the basis of counts from unique and multiple alignments using EdgeR within R ver. 3.2.2 (R Development Core Team, 2011) using BIOCONDUCTOR ver. 3.0. The alignment file
was also used for assembling transcripts, estimating their abundance, and detecting differential expression of genes or isoforms using cufflinks. We used the FPKM (fragments per kilobase of exon per million fragments) as the method of determining the expression level of the gene regions. The global normalization method was used for comparison between samples. Gene classification was based on searches done by DAVID (http://david.abcc.ncifcrf.gov/).

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Manufacturer protocol

Download the product protocol from Lexogen for SENSE mRNA-Seq Library Prep Kit V2 below.

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