NEBNext® Multiplex Small RNA Library Prep Set for Illumina®

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Starting Material: 100 ng–1 μg Total RNA. Small RNA fragments should have a 5 ́ phosphate and 3 ́ OH to ligate and must be free of ATP.

Protocol tips
Starting Material: 100 ng–1 μg Total RNA. Small RNA fragments should have a 5 ́ phosphate and 3 ́ OH to ligate and must be free of ATP.

Publication protocol

Total RNA was purified from ES cells using TRIzol Reagent (ThermoFisher Scientific), before treating with DNase Turbo (Ambion). RNA was then purified into mRNA and short and long RNA using Oligotext mRNA Mini Kit (Qiagen) and mirVana™ miRNA Isolation Kit (ThermoFisher Scientific), respectively. ERCC RNA Spike-In Mix (ThermoFisher Scientific) was added to all RNA samples. Post-purification, short RNAs were treated with Ribo-Zero (Illumina) to remove rRNA. Post purification, mRNAs were fragmented by magnesium treatment followed by phosphatase-mediated repair of cleaved ends using Antarctic Phosphatase (NEB) and T4 Polynucleotide Kinase (PNK) (NEB). Following a final cleanup using RNeasy Kit (Qiagen), sequencing libraries were generated for both mRNA and short RNA fractions using NEBNext Multiple Small RNA Library Prep Protocol (NEB). Depending on the starting amount of input RNA, between 10 and 12 cycles were used for amplification, followed by size fractionation by gel electrophoresis. Amplicons in the 130–350 bp size range were gel-excised and purified using Mini-Elute Gel Extraction Kit (Qiagen), followed by real-time PCR (qPCR)-based quantification using Kapa Library Quantification Kit (Kapa Biosystems) and analysis of size distribution using a Bioanalyzer High Sensitivity DNA Chip (Agilent). Samples were sequenced on a NextSeq 500 (Illumina).

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Manufacturer protocol

Download the product protocol from New England BioLabs for NEBNext® Multiplex Small RNA Library Prep Set for Illumina® below.

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