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

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	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 from each group of BV-2 cells i.e., control 2 h (2 samples), control 4 h (2 samples), LPS 2 h (2 samples), LPS 4 h (2 samples), Poly (I:C) 2 h (2 samples) and Poly (I:C) 4 h (2 samples), using TRIzol® (Life Technologies) according to the manufacturer’s instructions. For RNA-Seq, RNA libraries were created from each group of BV-2 cells using a NEBNext® Ultra™ Directional RNA Library Preparation Kit from Illumina®. The first step in the workflow involved the removal of ribosomal RNA using an RNAMius™ Transcriptome Isolation Kit (Life Technologies). Following purification, total RNA was fragmented into small pieces using divalent cations at elevated temperatures. The cleaved RNA fragments were subjected to first-strand cDNA synthesis using reverse transcriptase and random primers, followed by second-strand cDNA synthesis using DNA polymerase I and RNase H. The cDNA fragments were subsequently processed using an end-repair reaction after the addition of a single ‘A’ base, followed by adapter ligation. The products of these reactions were purified and enriched through PCR to generate the final cDNA library. The cDNA fragments were sequenced using an Illumina HiSeq2500 System (101 cycles, paired-end (PE) lane) (National Instrumentation Center for Environmental Management in Seoul National University). Biological duplicate RNA sequencing was performed on twelve independent RNA samples from the treated BV-2 cells.

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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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