The 5′-end of the RNA was probed using dRNA-Seq, as previously described (5 (link)). Briefly, rRNA-subtracted RNA was split into two samples. One sample was treated with 20 U RNA 5′ polyphosphatase (Epicentre, Madison, WI, USA) at 37°C for 60 min. The other sample was treated with nuclease-free water. The dephosphorylated RNA adaptor was ligated to both samples using 5 U of T4 RNA ligase (Epicentre) at 37°C for 90 min. The adaptor-ligated RNA samples were purified, and cDNA was synthesized using the SuperScript III First-Strand Synthesis System with 3.125 pmol random nonamers. DNA libraries were amplified by PCR with Phusion High-Fidelity DNA Polymerase, using P5 and P7 index primers for 20 cycles. The amplified libraries were sequenced via the 50 cycles single-ended recipe on a HiSeq 2500 sequencer. The oligonucleotide sequences are summarized in Supplementary Table S1 .
Protocol detail
5'-end RNA Probing via dRNA-Seq
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Cdna
Dna libraries
Dna polymerase
Oligonucleotide
Polyphosphatase
Primers
Rna ligase
Rrna
Synthesis
Corresponding Organization :
Other organizations : University of California, San Diego, Korea Advanced Institute of Science and Technology, Korea Research Institute of Bioscience and Biotechnology
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Variable analysis
independent variables
- Whether the RNA sample was treated with 20 U RNA 5' polyphosphatase at 37°C for 60 min or treated with nuclease-free water
- The sequencing of the adaptor-ligated RNA samples
- The concentration of the dephosphorylated RNA adaptor (5 U of T4 RNA ligase used for 90 min at 37°C)
- The cDNA synthesis using the SuperScript III First-Strand Synthesis System with 3.125 pmol random nonamers
- The DNA library amplification by PCR with Phusion High-Fidelity DNA Polymerase, using P5 and P7 index primers for 20 cycles
- The sequencing of the amplified libraries via the 50 cycles single-ended recipe on a HiSeq 2500 sequencer
- The control sample was treated with nuclease-free water instead of 20 U RNA 5' polyphosphatase.
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