NEBNext® Ultra™ RNA Library Prep Kit for Illumina®

Protocol tips

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	Total RNA (10 ng–1 μg) quantified by Bioanalyzer.

Protocol tips
Total RNA (10 ng–1 μg) quantified by Bioanalyzer.

Publication protocol

20 μl at 100 ng/μl of 3 independent biological replicate RNA samples isolated from separate passages of MDA-MB-231 wild-type and SH-4-54-resistant clone #2 cells, as well as triplicate independent biological replicates of T47D wild-type and SH-4-54-resistant clone #1 RNA samples, were submitted for RNA-sequencing (Farncombe Metagenomics Facility, McMaster University), also referred to as NextGeneration sequencing. RNA quality was validated using the RNA 6000 Nano kit and a 2100 Bioanalyzer (Agilent Technologies). A RNA library was prepared using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina, with the Next Poly(A) mRNA Magnetic Isolation Module (New England Biolabs) used to enrich poly-A mRNA. Each library was sequenced on the Illumina HiSeq 1500 platform (Illumina) via HiSeq Rapid V2 chemistry with onboard cluster generation and 70 bp single-end reads. Each biological replicate was split between two lanes to mitigate lane effects, with reads being subsequently combined during analysis using the Tuxedo protocol as previously described. The RNA-sequencing dataset was submitted to the National Institutes of Health (NIH) Sequence Read Archive (SRA) as SRA accession SRP078574.

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Manufacturer protocol

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

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