GSM1687554: IMR90_RDIP; Homo sapiens; OTHER
Whole cell nucleic acid was isolated through a modified salting out extraction protocol.  Nucleic acid was sonicated to an average size of 400-600 bp using the Covaris sonicator.  The fragmented nucleic acid was then treated with RNase I (Ambion AM2294) to remove any ssRNA from the sample, phenol/chloroform purified and re-suspended in EB buffer. Libraries were prepared using elements of a directional RNA-seq protocol.  Starting the library preparation at the second strand synthesis step, the RNA of the RNA:DNA hybrid was nicked using RNase H treatment to serve as a primer for the DNA polymerase.  The second strand was formed while incorporating dUTP to allow for directional sequencing and the identification of the RNA strand of the RNA:DNA hybrid.  Next, the ends of fragments were repaired, adenosine tails added, and Illumina Tru-Seq strand-specific adaptors ligated.  UNG treatment was utilized to degrade the dUTP-containing RNA strand of the RNA:DNA hybrid, and barcoded PCR primers were used to amplify the library while maintaining directionality.  Libraries were multiplexed and combined for sequencing using Illumina HiSeq 2500 150 bp paired-end sequencing in our institutional Epigenomics Shared Facility.
GEO Accession
GSM1687554