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Uniformity of Coverage across ERCC spike-ins
Dir_RNA_Transcript_Figure6
Poly(A)-containing mRNA was isolated from Universal Human Reference RNA (Agilent #740000) with recommended amounts of ERCC RNA Spike-In Mix I (Thermo Fisher Scientific #4456740), and libraries were made using the NEBNext Ultra II Directional RNA Kit (plus the NEBNext Poly(A) mRNA Magnetic Isolation Module), Illumina TruSeq Stranded mRNA Kit, Kapa Stranded mRNA-Seq Kit and Kapa mRNA HyperPrep Kit. Libraries were sequenced on an Illumina NextSeq® 500 using paired-end mode (2x76 bp). A global view of the transcript coverage of spiked-in ERCCs was determined by mapping reads directly to the ERCC sequences and assessing coverage using bedtools in 100 bins along the transcript length. Superior consistency of coverage between the range of library preparation input amounts (10 ng, 100 ng, 1 µg) was seen with the Ultra II Directional RNA Kit.

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Available Kits:

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NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina®
Includes optimized mixes for directional RNA library preparation (fragmentation, cDNA synthesis, end repair/dA-tailing, adaptor ligation and PCR enrichment steps) for sequencing on the Illumina platform.
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NEBNext® Ultra™ II Directional RNA Library Prep with Sample Purification Beads
Includes optimized mixes for directional RNA library preparation (fragmentation, cDNA synthesis, end repair/dA-tailing, adaptor ligation and PCR enrichment steps) plus SPRIselect® beads for size selection and cleanup.