AmpliSeq™ libraries were constructed and sequenced on the Ion Torrent Proton platform (Thermo Fisher) according to the manufacturer’s instructions as previously described24 (link),25 (link). For human gene analysis in cell lines, the Ion AmpliSeq Transcriptome Human Gene Expression Kit is designed for targeted amplification of over 20,000 human RefSeq genes simultaneously in a single primer pool. A short amplicon (~110 bp) is amplified for each targeted gene. Since an AmpliSeq transcriptome mouse kit is not commercially available, an Ion AmpliSeq™ Custom Panel was designed by the manufacturer (Thermo Fisher) using Ion AmpliSeq™ Designer for targeted amplification of 3,826 mouse genes that are most relevant for our studies (one short amplicon for each gene) in one primer pool for mouse studies. For each sample, 10 ng of total RNA was used for cDNA library preparation. Multiple libraries were multiplexed and clonally amplified using the Ion OneTouch 2 System (Thermo Fisher), and then sequenced on an Ion Torrent Proton machine (Thermo Fisher). Data was first analyzed by Torrent Suite and ampliSeqRNA analysis plugin (Thermo Fisher) to generate count data.
Protocol detail
AmpliSeq Transcriptome Profiling for Cell Lines
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Ion Torrent Proton platform
Ion AmpliSeq™ Custom Panel
Ion AmpliSeq™ Designer
Ion OneTouch 2 System
Ion Torrent Proton machine
Torrent Suite
ampliSeqRNA analysis plugin
Cdna library
Cell lines
Gene
Gene expression
Genes amplification
Human
Mouse
Primer
Proton
Transcriptome
Corresponding Organization : Broad Institute
Other organizations : Washington University in St. Louis, Baylor College of Medicine, Center for Systems Biology
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Variable analysis
independent variables
- Transcriptome sequencing kit used (Ion AmpliSeq Transcriptome Human Gene Expression Kit or Ion AmpliSeq™ Custom Panel)
- Gene expression levels
- Amount of total RNA used for cDNA library preparation (10 ng)
- Clonal amplification of libraries using the Ion OneTouch 2 System
- Sequencing platform (Ion Torrent Proton)
- No positive or negative controls were explicitly mentioned in the provided information.
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