Transcriptomic analysis of Thermoanaerobacter tengcongensis grown at different temperatures by RNA sequencing
A total amount of 3ug RNA per sample was used as input material for the RNA sample preparations. All four samples had RIN(RNA Integrity number) values above 8. Sequencing libraries were generated using NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, Ispawich, USA) following manufacturer’s recommendations and four index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads (Life technologies, CA, USA). Fragmentation was carried out using divalent cations under elevated temperature in NEB proprietary fragmentation buffer. First strand cDNA was synthesized using random oligonucleotides and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using dNTPs mixture containing dUTP, DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities and enzymes were removed. After adenylation of 3’ ends of DNA fragments, NEBNext adaptor oligonucleotides were ligated to cDNA fragments. In order to select cDNA fragments of preferentially 200 bp in length the library fragments were purified with AMPure XP beads system (Beckman Coulter, Beverly, USA). The index was introduced by PCR using NEB Universal PCR Primer and Index primer. The second strand cDNA that contain dUTP was digested by USER enzyme (NEB, Ispawich, USA). The first strand DNA fragments with ligated adaptor molecules on both ends were selectively enriched in a 10 cycles PCR reaction. Products were purified (AMPure XP beads system) and quantified using the Agilent high sensitivity DNA assay on the Agilent Bioanalyzer 2100 system.