GSM1655401: 16h_WT+_total; mixed sample; RNA-Seq
miRvana for total RNA (Ambion) miRvana kit for total RNA (Ambion) or TRIzol (Invitrogen) as indicated cDNA libraries for Illumina sequencing were generated by Vertis Biotechnology AG, Freising-Weihenstephan, Germany (http://www.vertisbiotech.com/). For the total RNA sample ~500 ng were used for cDNA library preparation. DNase I-treated total RNA samples were first sheared via ultra-sound sonication (4 pulses of 30 s at 4°C each) to generate on average ~200-400 bp fragmentation products. Then, fragments <20 nt were removed using the Agencourt RNAClean XP kit (Beckman Coulter Genomics). The samples were then poly(A)-tailed using poly(A) polymerase and the 5’triphosphate (and eukaryotic cap) structures were removed using tobacco acid pyrophosphatase (TAP). Afterwards, an RNA adapter was ligated to the 5’monophosphate of the RNA. First-strand cDNA synthesis was performed using an oligo(dT)-adapter primer and the M-MLV reverse transcriptase (NEB). The cDNA library for the rRNA-depleted sample was constructed similarly, except for the following modifications in the protocol: Upon the fragmentation of the RNA, a dephosphorylation step with Antarctic Phosphatase (AP, NEB) and a re-phosphorylation with T4 Polynucleotide Kinase (PNK, NEB) were performed. Oligonucleotide adapters were ligated to both the 5' and 3' end of the RNA samples. First-strand cDNA synthesis was performed using M-MLV reverse transcriptase and the 3’ adapter as primer. In any case, the resulting cDNA was PCR-amplified to about 20-30 ng/µL using a high fidelity DNA polymerase (barcode sequences for multiplexing were part of the 3’primers). The cDNA library was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and analyzed by capillary electrophoresis (Agilent Technologies).
GEO Accession
GSM1655401