TruSeq Stranded Total RNA

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	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

RNA purity was determined using a NanoDrop8000 spectrophotometer. RNA integrity was assessed using an 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) with an RNA Integrity Number value. Total RNA sequencing libraries were prepared according to the manufacturer’s instructions using a Truseq Stranded Total RNA Sample prep kit (Illumina, San Diego, CA, USA). One microgram of total RNA was further treated for rRNA depletion using Ribo-zero rRNA Removal Kit (human/mouse/rat) (Illumina). After purification, the rRNA-depleted total RNA was fragmented into small pieces using divalent cations under 94°C for 8 min. Cleaved RNA fragments were copied to first strand cDNA using reverse transcriptase and random primers. This was followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. Finally, single ‘A’ base was added and subsequent ligation of the adapter was performed. The products were purified and enriched with PCR to create the final cDNA library. The quality of the amplified libraries was verified by capillary electrophoresis (Agilent). After QPCR using SYBR Green PCR Master Mix (Applied Biosystems, Waltham, MA, USA), index-tagged libraries were combined in an equimolar ratio. RNA sequencing was performed using the Illumina NextSeq 500 platform according to the manufacturer’s recommended protocol for 2x75 sequencing. We deposited the RNA sequencing (RNA seq) data set in the Sequence Read Archive of the National Center for Biotechnology Information (accession number PRJNA433563, SRP132517).

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Manufacturer protocol

Download the product protocol from Illumina for TruSeq Stranded Total RNA below.

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