NEBNext® Ultra™ RNA Library Prep Kit for Illumina®

Protocol tips

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

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

Publication protocol

Total RNA from cell lysates was isolated using Phol/chloroform. The quality and integrity of the total RNA were evaluated using the Agilent 2100 Bioanalyzer and Agilent RNA 6000 Nano Kit (Agilent Technologies, USA), respectively. Then mRNA was purified using oligo (dT) magnetic beads, and the high-quality mRNA samples from each group were pooled for preparation of cDNA library using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina®. The mRNA was fragmented into small pieces using an additional fragmentation buffer, then the first-strand cDNA was synthesized using random hexamer-primers. The cDNA was further converted into double-stranded cDNA which was subsequently purified with AMPure XP beads. The purified double-stranded cDNA was performed end repair, dA tailing and adaptor ligation. Size selection was performed using AMPure XP beads, generating cDNA libraries. The libraries were enriched using PCR. The paired-end libraries were then sequenced on the Illumina HiSeq 4000 platform, generating paired-end reads of 150 bp. The data are available at National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database under the accession number SRP078920.

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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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