GSM1439468: WT1; Danio rerio; RNA-Seq
RNA was extracted using TRIzol (Invitrogen) according to the manufacturer's instructions The library of template molecules suitable for high throughput DNA sequencing was created following the Illumina “mRNA sequencing sample preparation guide” (part #1004898 Rev.D) with some modifications. Briefly, mRNA was purified from 2 µg total RNA using oligo-dT magnetic beads and fragmented using divalent cations at 94°C for 5 minutes. The cleaved mRNA fragments were reverse transcribed to cDNA using random primers, then the second strand of the cDNA was synthesized using DNA Polymerase I and RNase H. The next steps of RNA-Seq Library preparation were performed in a fully automated system using SPRIworks Fragment Library System I kit (ref A84801, Beckman Coulter, Inc) with the SPRI-TE instrument (Beckman Coulter, Inc). Briefly, in this system, double stranded cDNA fragments were blunted, phosphorylated and ligated to indexed adapter dimers, and fragments in the range of ~200-400 bp were size selected. The automated steps were followed by PCR amplification (30 sec at 98°C; [10 sec at 98°C, 30 sec at 60°C, 30 sec at 72°C] x 13 cycles; 5 min at 72°C), then surplus PCR primers were removed by purification using AMPure XP beads (Agencourt Biosciences Corporation). DNA libraries were checked for quality and quantified using a 2100 Bioanalyzer (Agilent).
GEO Accession
GSM1439468