To arrive at this conclusion, the authors initially isolated total RNA from bone marrow cells, removed rRNA using the Ribo-Zero™ Kit (Human/Mouse/Rat), prepared RNA-Seq libraries using the Illumina TruSeq RNA-Seq Sample Prep Kit, and sequenced the libraries on an Illumina GAIIx sequencer. Leukemia samples were sequenced together, while remission blood samples and normal bone marrow subpopulations were sequenced in separate runs. In another analysis, the authors used paired-end mapping ratios to determine the relative abundance of scrambled isoforms to normal linear isoforms. Each gene was tiled by dividing it into even-length bins of 200 bp. Data were validated by qRT-PCR.
The researchers further concluded that the scrambled splicing activity appears to occur most commonly in the cytoplasm. Four potential models for circularization were presented, using techniques that also demonstrated the presence of circular RNA splice variants using RNase R exoribonuclease.
Salzman, J. et al. (2012). Circular RNAs Are the Predominant Transcript Isoform from Hundreds of Human Genes in Diverse Cell Types PLoS ONE, 7 (2) DOI: 10.1371/journal.pone.0030733