GSM8390076: TEX treatment, 33h, Replicate 2; Nocardia terpenica; RNA-Seq
Firstly, the four RNA samples R1-33h, R1-48h, R2-33h and R2-48h were treated with T4 polynucleotide kinase.  The RNA samples were then split into two halves and one half was subjected to Terminator™ 5'-phosphate-dependent exonuclease treatment (+TEX), the other half was left untreated (-TEX). For cDNA synthesis, the RNA samples were poly(A)-tailed to the 3'-end using poly(A) polymerase.  Then, the 5'-triphosphate residues were removed using RNA 5'-polyphosphatase. Accordingly, an RNA adapter was ligated to the 5'-monophosphate residue of the RNA. First strand cDNA synthesis was performed using an oligo(dT)-adapter primer and the M-MLV reverse transcriptase. The resultant cDNAs were PCR-amplified to approximately 10-20 ng/μl using DNA polymerase. Finally, the cDNAs were purified using an Agencourt AMPure XP kit and analyzed via capillary electrophoresis. For Illumina NextSeq sequencing, the eight cDNA samples were pooled in about equimolar amounts. Subsequently, the cDNA pool was eluted from a preparative agarose gel in the size range of 200-500 bp. The primers used for PCR amplification were designed for TruSeq sequencing according to the instructions of Illumina. Finally, the cDNA pool was single-end sequenced on an Illumina NextSeq 500 system using 1x75bp read length.