GSM5829013: texAM 1 [RNA-seq]; Mus musculus; RNA-Seq
RNA was extracted using the Qiagen miRNeasy Kit. Amplified cDNA was prepared from 2 ng of total RNA using the Ovation RNA-seq system V2 (NuGEN Technologies, Inc.) following manufacturer's instructions. Briefly, first-strand cDNA was prepared from total RNA using a combination of random and poly-T DNA/RNA chimeric SPIA primers and reverse transcriptase, resulting in a cDNA/mRNA hybrid molecule. Priming sites created by a heating fragmentation of the mRNA within the cDNA/mRNA complex were used to synthesize the second strand cDNA using a DNA polymerase. The resulting double-stranded cDNA with a unique RNA/DNA heteroduplex at one end was purified using Agencourt RNAClean XP beads (Beckman Coulter Inc.). The purified product was then used as the substrate of a linear amplification process, SPIA (single primer isothermal amplification). During the amplification step, RNase H degrades RNA in DNA/RNA heteroduplex at the 5'end of the first cDNA strand, creating a binding site for the DNA/RNA SPIA chimeric primer, then DNA polymerase starts replication at the 3'end of the primer displacing the existing forward strand. The process including SPIA DNA/RNA primer binding, DNA replication, strand displacement and RNA degradation is repeated and results in rapid generation of microgram quantities of amplified cDNA. Amplified cDNA was purified using AMPure XP beads (Beckman Coulter Inc.) and 500 ng was fragmented by sonication using a Covaris E210 instrument (with duty cycle: 10X, intensity: 5 and cycle/burst: 200 for 180 seconds). The next steps of RNA-Seq Library preparation were performed using Ovation Ultralow Library System kit (NuGEN Technologies, Inc.) according to manufacturer's instructions. Briefly, in this system 100 ng of amplified cDNA were blunted, phosphorylated and ligated to indexed adapter dimers. The libraries were then enriched by PCR amplification (2 min at 72 degrees Celsius; [30 sec at 94 degrees Celsius, 30 sec at 60 degrees Celsius, 1 min at 72 degrees Celsius] x 6 cycles; 5 min at 72 degrees Celsius) and surplus PCR primers were removed by purification using AMPure XP beads. DNA libraries were checked for quality using 2100 Bioanalyzer (Agilent) and quantified using Kapa Sybr Fast Light Cycler 480 qPCR Kit (Kapa Biosystems) following manufacturer's instructions.