GSM1354477: RD19-NI+TEX; Deinococcus deserti; RNA-Seq
RNAprotect-treated cells were centrifuged and cell pellets stored at -80°C. Cell pellets were pretreated with lysozyme and proteinase K. Total RNAs were isolated using the mirVana RNA isolation kit (Ambion) including DNase treatment. From each RNA preparation (i.e. non-irradiated and irradiated cells), two cDNA syntheses were carried out, one with and one without Terminator exonuclease (TEX, Epicentre) treatment. cf SAMPLES RNA was incubated with TEX (Terminator exonuclease (TEX, Epicentre)), which specifically degrades RNA species that carry a 5' mono-phosphate (5'P). The exonuclease resistant RNA (primary transcripts with 5'PPP) was poly(A)-tailed using poly(A) polymerase and treated with TAP (tobacco acid pyrophosphatase), which degrades 5'PPP to 5'P. Then an RNA adapter was ligated to the 5'P of the “de-capped” RNA. First-strand cDNA synthesis was performed using an oligo(dT)-adapter primer and M-MLV reverse transcriptase. The resulting cDNAs were PCR-amplified to about 30 ng/μl using a high fidelity DNA polymerase. The cDNAs were purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and analyzed by capillary electrophoresis. The primers used for PCR amplification were designed for TruSeq sequencing according to the instructions of Illumina, with the 3’-sequencing adapter containing a barcode specific for each library. The +TEX cDNA samples were pooled at approximately equimolar amounts and the cDNA pool was used for sequencing without further treatment. The pools were sequenced on a Illumina HiSeq 2000 machine (read length: 100 bp).
GEO Accession
GSM1354477