TruSeq RNA Library Prep Kit v2

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Ribosomal RNA in total RNA samples was removed before library construction using the Ribo-Zero rRNA Removal Kits (Epicentre Technologies, Madison, WI). Adapters containing seven nucleotide indexes were ligated to the double-stranded cDNA	Illumina RNAseq libraries were prepared with the TruSeq RNA Sample Prep kit (Illumina, San Diego, CA) per manufacturer’s protocol.

Upstream tips
Ribosomal RNA in total RNA samples was removed before library construction using the Ribo-Zero rRNA Removal Kits (Epicentre Technologies, Madison, WI). Adapters containing seven nucleotide indexes were ligated to the double-stranded cDNA
Protocol tips
Illumina RNAseq libraries were prepared with the TruSeq RNA Sample Prep kit (Illumina, San Diego, CA) per manufacturer’s protocol.

Publication protocol

Illumina RNAseq libraries were prepared with the TruSeq RNA Sample Prep kit (Illumina, San Diego, CA) per manufacturer’s protocol. Ribosomal RNA in total RNA samples was removed before library construction using the Ribo-Zero rRNA Removal Kits (Epicentre Technologies, Madison, WI). Adapters containing seven nucleotide indexes were ligated to the double-stranded cDNA. The DNA was purified between enzymatic reactions and the size selection of the library was performed with AMPure XT beads (Beckman Coulter Genomics, Danvers, MA). Libraries were assessed for concentration and fragment size using the DNA High Sensitivity Assay on the LabChip GX (Perkin Elmer, Waltham, MA). The library concentrations were also assessed by qPCR using the KAPA Library Quantification Kit (Complete, Universal) (Kapa Biosystems, Woburn, MA). The libraries were pooled and sequenced on a 100 base pairs paired-end Illumina HiSeq 2500 run (Illumina, San Diego, CA). The sequenced reads were aligned to the reference sequence (Ensembl version Rnor_5.0.77) using TopHat version 1.4.1 (Trapnell et al., 2009).21 The alignments allowed up to 2 bp mismatches per 25 bp segment, and we removed reads that aligned to more than 20 different genomic regions. Transcript abundances and splice variant identification was done using Cufflinks version 1.3 using the BAM alignment files obtained from TopHat.22 BigWig coverage files were generated from the BAM alignment files using the UCSC genome browser tools.

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Manufacturer protocol

Download the product protocol from Illumina for TruSeq RNA Library Prep Kit v2 below.

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