GSM1259076: A2780-pBABE; Homo sapiens; RNA-Seq
Total RNA was extracted using the miRNeasy kit (Qiagen)  from two polyclonal pools of A2780 cells expressing exogenous miR-181a (p181a #1 and p181a #2) and pBABE contro.l The Illumina RNASeq kit was used to prepare sequencing libraries. The method is as follows: 1 μg of high quality total RNA was incubated with oligo(dT) magnetic beads (SeraMag) in order to enrich for mRNA with poly-A tails. Eluted RNA was randomly cleaved by incubation at 94°C with a magnesium buffer. The RNA is ethanol-precipitated and then reverse transcribed (Invitrogen SuperScript II). DNA polymerase is added to generate a second strand. dsDNA after second strand synthesis contains ends with 5´ and 3´ overhangs, and these are filled in using T4 polymerase and T4 polynucleotide kinase resulting in a blunt-ended DNA molecule. Then a deoxyadenosine (dA) 5´ tail is added to DNA strands using the Klenow fragment (exo-). dsDNA adapters with 3´ thymidine overhangs are ligated to the dA-tailed library using T4 ligase. The library was then size selected on a 2% agarose gel, and amplified with 15 cycles of PCR (NEB Phusion polymerase). This procedure adds the sequences needed for binding to the Illumina flow cell and sequencing primer-binding sites. RNA-Seq libraries were sequenced using the Illumina HiSeq 2000 for a 100nt read length. Transcriptome raw reads from Illumina HiSeq 2000 were mapped to the human reference genome (hg19). The coverage over the length of the gene is variable due to various biases inherent to the sequencing and sample preparation process. To avoid problems arising from this, we generated a density profile using windows of 50 bp sliding over the length of each gene, and the median of this density profile was picked as the representative coverage for the gene using an in-house mRNA-seq expression profile pipeline. The coverage is corrected using log-transformation and quantile normalization.
GEO Accession
GSM1259076