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

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Starting Material: 100 ng to 1 μg total RNA in up to 12 μl total volume.

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

Publication protocol

Total RNA was extracted using an RNeasy Mini Kit (Qiagen). RNA quantification and quality checks were performed with a Quantus fluorometer (Promega) and the Bioanalyzer RNA 6000 Nano Kit (Agilent Technologies). We confirmed that the sample RNA integrity number was >9.5.
Bulk RNA-seq
For paRNA-seq, we purified poly(A) RNA from 1 µg of ES total RNA using a NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB). For rdRNA-seq, we depleted rRNA from 1 µg of ES total RNA using a GeneRead rRNA Depletion Kit (Qiagen). We prepared conventional RNA-seq library DNA with the resulting RNA using a commercial kit (NEBNext Ultra Directional RNA Library Prep Kit for Illumina; NEB) in accordance with the manufacturer’s protocol, with slight modifications during RT and PCR steps. We used SuperScript III (Thermo Fisher) for RT. We used KAPA HiFi DNA polymerase (Kapa Biosystems) in the PCR step. When we performed rdRNA-seq using 10 or 1 ng of ES total RNA, we utilized a KAPA RNA HyperPrep Kit with RiboErase (Kapa Biosystems) for the preparation of sequencing library DNA according to the manual instructions.

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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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