Differential RNA-seq (dRNA-seq) of Pyrococcus furiosus
RNA purified from cells grown under eight different growth conditions were pooled equally and submitted for library preparation and sequencing to the Core Unit Systems Medicine (SysMed) at the University Wrzburg, Germany. Three different libraries were prepared to fulfill the requirements for usage in the ANNOgesic pipeline: fragmented, unfragmented with terminator exonuclease treatment (+TEX) and unfragmented without TEX-treatment (-TEX). For the fragmented sample, RNA was fragmented for 2 min at 94 C using the NEBNext Magnesium RNA Fragmentation Module. Afterwards RNA was treated with T4 Polynucleotide Kinase (PNK) without ATP for 6 h at 37 C and 1 h at 37 C with 2 mM ATP and fresh T4 PNK. After overnight ethanol precipitation, 5 triphosphates were removed using RNA 5' Pyrophosphohydrolase (RppH) for 1 h at 37 C. RNA was again precipitated and resuspended in 6 l H2O. The two samples (+/- TEX) for the transcription start site detection were either treated with TEX (+ TEX) or with H2O as a mock control (- TEX) for 30 min at 37 C. Afterwards both samples were treated with RppH for 1 h at 37 C before they were precipitated and the RNA was resuspended in 6 l H2O. After the pre-treatment, all three libraries were prepared using the NEBNext Multiplex Small RNA Library Prep Kit for Illumina according to the manufacturers protocol with small modifications. The first linker ligation was performed for 18 h at 16 C and libraries were amplified with 12 PCR cycles with an extended elongation time of 75 s. Libraries were pooled in a 2:1:1 ratio (fragmented : + TEX : - TEX) and sequenced on an Illumina NextSeq 500 high-output single-end 75 nt run.