GSM1655373: 24 h WT GFP+ R3; 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/). A minimal amount of ~100 ng of total RNA was required 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). As an internal quality control for this pilot experiment, spike-in RNA (sequence of spike: 5’-AAAUCCGUUCGUACGGGCCC-3’; was 5’-monophosphorylated and gel-purified) was added to a final concentration of 0.5%. 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 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
GSM1655373