The RACE protocol was performed as described (45 (link)). Briefly, total RNA was extracted using TRIzol (Ambion), followed by ligation of 5′-adenylated-3′-blocked adapter (New England Biolabs) using T4 RNA ligase truncated KQ (New England Biolabs). Ligated RNAs were purified and cDNAs were synthesized using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific). PCR amplification was performed using gene specific primers and PCR amplicons were resolved on 2.5% agarose gels to visualize mature and extended transcripts. Primers used are given in Supplementary Table S1 .
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RACE protocol for transcriptome analysis
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Corresponding Organization :
Other organizations : Boston Children's Hospital, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Harvard University, Tufts University, University of Michigan–Ann Arbor
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Variable analysis
independent variables
- Use of TRIzol (Ambion) for total RNA extraction
- Ligation of 5′-adenylated-3′-blocked adapter (New England Biolabs) using T4 RNA ligase truncated KQ (New England Biolabs)
- CDNA synthesis using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific)
- PCR amplification using gene specific primers
- Visualization of mature and extended transcripts by resolving PCR amplicons on 2.5% agarose gels
- Primer sequences used for PCR amplification (provided in Supplementary Table S1)
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