GSM1354475: RD19-NI; 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 directly poly(A)-tailed using poly(A) polymerase, followed by TAP treatment. An RNA adapter was then ligated to the 5'P of the total RNA samples. First-strand cDNA synthesis was performed using an oligo(dT)-adapter primer and M-MLV H- reverse transcriptase. The resulting cDNAs were PCR-amplified to about 60-90 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. For Illumina sequencing, the samples were pooled at approximately equimolar amounts and size fractionated in the range between 250 – 500 bp on Agarose Gel. The pools were sequenced on a Illumina HiSeq 2000 machine (read length: 100 bp).
GEO Accession
GSM1354475