In a subset of human samples, carrier poly(rA) RNA and host rRNA were depleted from RNA samples using RNase H selective depletion9 (link),33 (link). In brief, oligo d(T) (40 nt long) and/or DNA probes complementary to human rRNA were hybridized to the sample RNA. The sample was then treated with 15 units Hybridase (Epicentre) for 30 min at 45 °C. The complementary DNA probes were removed by treating each reaction with an RNase-free DNase (Qiagen) according to the manufacturer’s protocol. Following depletion, samples were purified using 1.8× volume AMPure RNAclean beads (Beckman Coulter Genomics) and eluted into 10 µ l water for cDNA synthesis.
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Other organizations :
Broad Institute, Center for Systems Biology, Harvard University, Florida Gulf Coast University, Miami-Dade Public Library System, Florida Department of Health, University of the West Indies System, National Autonomous University of Honduras, Industrial University of Santander, University of Florida, Massachusetts Department of Public Health, Scripps Research Institute, University of Edinburgh, Massachusetts Institute of Technology, Fred Hutch Cancer Center, University of Córdoba, Howard Hughes Medical Institute
Hybridization of oligo d(T) (40 nt long) and/or DNA probes complementary to human rRNA to the sample RNA
Treatment with 15 units Hybridase (Epicentre) for 30 min at 45 °C
dependent variables
Depletion of carrier poly(rA) RNA and host rRNA from RNA samples
control variables
Removal of complementary DNA probes by treating each reaction with an RNase-free DNase (Qiagen) according to the manufacturer's protocol
controls
Positive control: Not specified
Negative control: Not specified
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