Nebnext ultra 2 directional dna library prep kit

Genomic DNA was isolated using the phenol extraction method [4 ]. Sequencing was done by Eurofins Genomics Europe Sequencing Gmbh (Konstanz, Germany) with NovaSeq 6000 platform and 150 bp paired‐end configuration. For library preparation, TruSeq Adapter sequences were utilized using a self‐validated and established protocol of Eurofins Genomics, based on NEBNext Ultra II Directional DNA Library Prep Kit for Illumina. Default parameters were used in the following analysis tools. The quality control for the 3,651,578 Illumina raw reads was done with FastQC v.0.11.3 [17 ].

ChIP‐seq and polyA‐selected total RNA libraries were prepared with NEBNext® Ultra II DNA Library Prep Kit for Illumina. Optimum PCR cycles were determined via StepOnePlus qPCR using PowerTrack SYBR Green reagents (Thermo Scientific). Ribo‐zero‐selected total RNA libraries were prepared with NEBNext® Ultra II Directional DNA Library Prep Kit for Illumina.
ChIP‐seq, 5′ dependent and independent small RNA, and polyA‐selected total RNA libraries were sequenced on a HiSeq 1500 machine at the Gurdon Institute.
Total RNA was extracted using TRIzol reagent (Ambion, Life Technologies) and treated with Turbo DNase Kit (Invitrogen) according to the manufacturer’s instructions. 1 μg total RNA was used for library preparation. Ribo‐zero‐selected total RNA libraries were sequenced on a HiSeq 2500 at the Cancer Institute, Cambridge. All sequencing was performed with SE50.
Short capped RNA sequencing from nucleoplasmic and chromatin gradients were sequenced at the LMS Sequencing Facility on a HiSeq 2500 with SE75.