GSM2098937: KO_rep3; Mus musculus; RNA-Seq
Total RNA was extracted using Tri Reagent (Molecular Research Center, TR118) Libraries of template molecules suitable for high throughput DNA sequencing were created using “TruSeq™ Stranded Total RNA LT library prep kit with Ribo-Zero Gold” (Illumina Inc.) according to manufacturer’s user guide (Rev E oct 2013). Briefly, 300 ng of total RNA were depleted in ribosomal RNA using biotinylated, target-specific oligos combined with Ribo-Zero™ removal beads. Following purification, RNA were fragmented using divalent cations at 94°C for 8 minutes. The cleaved RNA fragments were then reverse transcribed to cDNA using random primers and the second strand of cDNA was synthesized using DNA Polymerase I and RNase H. The double stranded cDNA fragments were blunted using T4 DNA polymerase, Klenow DNA polymerase and T4 PNK. A single ‘A’ nucleotide was added to the 3’ ends of the blunt DNA fragments using a Klenow fragment (3' to 5'exo minus) enzyme. The cDNA fragments were ligated to double stranded adapters using T4 DNA Ligase. The ligated products were enriched by PCR amplification (30 sec at 98°C; [10 sec at 98°C, 30 sec at 60°C, 30 sec at 72°C] x 12 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 2100 Bioanalyzer (Agilent). The libraries were loaded in the flow cell at 7pM concentration and clusters were generated in the Cbot and sequenced on the Illumina Hiseq2500 as single-end 50 base reads following Illumina’s instructions.
GEO
GSM2098937