NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina®

Protocol tips

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	100 ng to 1 μg total RNA in up to 12 μl total volume.

Protocol tips
100 ng to 1 μg total RNA in up to 12 μl total volume.

Publication protocol

Total RNA was extracted using RNAiso Plus (Takara Bio Inc., Shiga, Japan) and a QIAGEN RNeasy® Mini kit (QIAGEN, Hilden, Germany). BMDMs or RAW264.7 macrophage cell line was completely lysed using RNAiso Plus and then 200 µl of chloroform was added. The tubes were then inverted for 5 min. The mixture was centrifuged at 12,000 × g for 15 min at 4°C, and the upper phase was placed into a new tube. A 600 µl volume of 70% ethanol was added and the mixture was applied to an RNeasy mini column. The column was washed with wash buffer. To elute the RNA, RNase-free water (30 µl) was added directly onto the RNase mini column, which was then centrifuged at 12,000 × g for 3 min at 4°C. To deplete ribosomal RNA (rRNA) from the total RNA preparations, a RiboMinus Eukaryote kit (Life Technologies, Carlsbad, CA, USA) was used according to the manufacturer’s instructions. RNA libraries were created using a NEBNext® Ultra™ directional RNA library preparation kit for Illumina® (New England Biolabs, Ipswich, MA, USA). The obtained rRNA-depleted total RNA was fragmented into small pieces using divalent cations at elevated temperatures. First-strand cDNA was synthesized using reverse transcriptase and random primers and second-strand cDNA synthesis was then performed using DNA polymerase I and RNase H. The cDNA fragments were processed using an end-repair reaction after the addition of a single “A” base, followed by adapter ligation. These products were purified and amplified using PCR to generate the final cDNA library. The cDNA fragments were sequenced using an Illumina HiSeq2000.

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Manufacturer protocol

Download the product protocol from New England BioLabs for NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® below.

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