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

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Starting Material: 100 ng-1μl total RNA in a 12 μl total volume.

Protocol tips
Starting Material: 100 ng-1μl total RNA in a 12 μl total volume.

Publication protocol

Total RNAs of control and corticosterone treated PC12 cells were extracted by using TRIzol
(Life Technologies, Grand Island, NY, USA). The quantity and quality of extracted RNAs were evaluated using NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA), Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara CA, USA), and RNA electropherograms with calculation of RNA integrity number (RIN), respectively (Schroeder et al. 2006). RNA samples were subjected to next
generation RNA-sequencing if their RIN > 7 and total RNA quantity > 400 ng. Six sequencing libraries (3 replicates for both control and corticosterone treatment groups) were constructed by using NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA) with poly (A) messenger RNA selection. The libraries were then used to build clusters on the Illumina flow cell, according to manufacturer protocol. Sequencing was performed using Illumina HiSeq 2500 System (Illumina, San Diego, CA, USA) with a 125 bp paired-end sequencing strategy.

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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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