Total RNA was purified using an RNeasy Micro Kit (Qiagen) and treated with the DNase-one Kit (Qiagen) to remove genomic DNA. Briefly, 10 ng of total RNA was transcribed to obtain single-stranded cDNA using the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific). A cDNA library was synthesized using the Ion AmpliSeq Transcriptome Human Gene Expression Core Panel (Thermo Fisher Scientific) and Ion Ampliseq Library Kit Plus (Thermo Fisher Scientific) according to the manufacturer’s protocol. Barcode-labeled cDNA libraries were analyzed by the Ion S5 XL System (Thermo Fisher Scientific) using the Ion 540 Chip Kit (Thermo Fisher Scientific). Total RNA was purified using the RNeasy Micro Kit (Qiagen) and treated with the DNase-one Kit (Qiagen) to remove genomic DNA. We reverse transcribed 10 ng of total RNA to obtain single-stranded cDNA using the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific). We performed cDNA library synthesis using the Ion AmpliSeq Transcriptome Human Gene Expression Core Panel (Thermo Fisher Scientific) and the Ion Ampliseq Library Kit Plus (Thermo Fisher Scientific) according to the manufacturer’s protocol. Barcode-labeled cDNA libraries were analyzed by the Ion S5 XL System (Thermo Fisher Scientific) using the Ion 540 Chip Kit (Thermo Fisher Scientific).
Ion ampliseq transcriptome human gene expression core panel
Manufactured by Thermo Fisher Scientific
The Ion AmpliSeq Transcriptome Human Gene Expression Core Panel is a targeted gene expression panel that allows for the analysis of approximately 20,000 coding transcripts from the human genome. The panel utilizes Ion AmpliSeq technology to provide a comprehensive overview of gene expression levels in a single assay.
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Lab products found in correlation
Ion ampliseq transcriptome human gene expression kit, by Thermo Fisher Scientific (4 mentions)
Ion xpress barcode adapter, by Thermo Fisher Scientific (4 mentions)
Ion pi hi q sequencing 200 kit, by Thermo Fisher Scientific (3 mentions)
Ion 540 chip kit, by Thermo Fisher Scientific (3 mentions)
Ion ampliseq library kit plus, by Thermo Fisher Scientific (3 mentions)
Ion s5 xl system, by Thermo Fisher Scientific (3 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (3 mentions)
Rneasy micro kit, by Qiagen (3 mentions)
Ion pi chip v3, by Thermo Fisher Scientific (3 mentions)
Agencourt ampure xp reagent, by Beckman Coulter (2 mentions)
Dnase one kit, by Qiagen (2 mentions)
Dna clean concentrator 5, by Zymo Research (2 mentions)
Ion pi hi q ot2 200 kit, by Thermo Fisher Scientific (2 mentions)
Ion pi hi q chef kit, by Thermo Fisher Scientific (1 mentions)
Ion ampliseq transcriptome human gene expression kit preparation, by Thermo Fisher Scientific (1 mentions)
Agilent bioanalyzer instrument, by Agilent Technologies (1 mentions)
Agilent high sensitivity dna kit, by Agilent Technologies (1 mentions)
Ion library taqman quantitation kit, by Thermo Fisher Scientific (1 mentions)
Torrent suite software, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
8 protocols using ion ampliseq transcriptome human gene expression core panel
1
Transcriptome Analysis by Ion AmpliSeq
2
Single-Cell Transcriptome Analysis of Fibroblasts
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Total RNA extracted from fibroblast cells was reverse transcribed according to Ion AmpliSeq™ Transcriptome Human Gene Expression kit Preparation protocol (Revision A.0; Thermo Fisher Scientific). The cDNA was amplified using Ion AmpliSeq™ Transcriptome Human Gene Expression core panel (Thermo Fisher Scientific), and the primer sequences were then partially digested, before ligation of Ion P1 Adapters and Ion Xpress™ Barcode Adapters (Thermo Fisher Scientific). Adaptor‐ligated amplicons were purified using Agencourt® AMPure® XP reagent (Beckman Coulter) and eluted in amplification mix (Platinum® PCR SuperMix High Fidelity and Library Amplification Primer Mix; Thermo Fisher Scientific) and amplified. Size selection and purification were conducted using Agencourt® AMPure® XP reagent (Beckman Coulter). The amplicons were quantified using Agilent® Bioanalyzer™ instrument with Agilent® High Sensitivity DNA kit (Agilent).
Samples were then pooled five and five, followed by template preparation on the Ion Chef system using the Ion PI Hi‐Q Chef Kit (Thermo Fisher Scientific). Samples were sequenced on the Ion Proton™ system using the Ion PI Hi‐Q Sequencing 200 Kit on Ion PI v3 chips (200 bp read length; Thermo Fisher Scientific).
Samples were then pooled five and five, followed by template preparation on the Ion Chef system using the Ion PI Hi‐Q Chef Kit (Thermo Fisher Scientific). Samples were sequenced on the Ion Proton™ system using the Ion PI Hi‐Q Sequencing 200 Kit on Ion PI v3 chips (200 bp read length; Thermo Fisher Scientific).
3
Ampliseq Transcriptome Library Preparation
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Total RNA was purified using the RNeasy Micro Kit (Qiagen) and treated using the DNase-one kit (Qiagen) to remove genomic DNA. We reverse transcribed 10 ng of total RNA to obtain single-stranded cDNA using the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific). We performed cDNA library synthesis for the Ion Ampliseq transcriptome using the Ion AmpliSeq Transcriptome Human Gene Expression Core Panel (Thermo Fisher Scientific) and the Ion Ampliseq Library Kit Plus (Thermo Fisher Scientific) according to the manufacturer’s protocol. Briefly, cDNA was amplified 12 cycles with Ion AmpliSeqTM Transcriptome Human Gene Expression Core Panel by thermal cycler. Primer sequences were partially digested with FuPa reagent by sequentially performing 10 min at 50 °C, 10 min at 55 °C, 20 min at 60 °C. Barcode ligation was performed with Ion Xpress Barcode for 30 min at 22 °C. Barcode-labeled cDNA libraries were purified by DNA Clean & Concentrator™-5 (Zymo research, CA, USA) and analyzed using the Ion S5 XL System (Thermo Fisher Scientific) and the Ion 540 Chip Kit (Thermo Fisher Scientific).
4
Targeted Gene Expression Profiling
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A total of 100 ng total RNA was reverse transcribed using the Ion AmpliSeq Transcriptome Human Gene Expression Kit following the manufacturer’s protocol (Thermo Fisher Scientific). Target genes (SQSTM1, MAP1LC3A, PINK1, MRPS16, MRPS9, and TOMM22) were amplified using an Ion AmpliSeq Transcriptome Human Gene Expression Core Panel (Thermo Fisher Scientific). Amplicons were ligated to barcode adapters and purified using Agencourt AMPure XP reagent (Beckman Coulter Inc). After purification, amplicons were eluted and normalized before emulsion PCR and chip loading on the Ion Chef system, obtaining reads with an average length of 200 bp. The sequencing data were aligned using the ampliSeqRNA plugin for Ion Torrent sequencing platforms (Thermo Fisher Scientific).
5
Transcriptome Profiling of Multiple Myeloma Cell Lines
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Total RNA was isolated and purified from KMS-11, KMS-20, KMS-26, KMS-27, KMS-28BM, MM.1R, MM.1S, NCI-H929, RPMI8226, and U266B1 cells using an RNeasy Mini kit (Qiagen, Valencia, CA, USA). A total of 10 ng RNA was reverse transcribed using the Ion AmpliSeq Transcriptome Human Gene Expression kit (Thermo Fisher Scientific) following the manufacturer's protocol. cDNA libraries were amplified and barcoded using Ion AmpliSeq Transcriptome Human Gene Expression core panel and Ion Xpress Barcode Adapter (Thermo Fisher Scientific). The prepared libraries were purified using Agencourt AMPure XP (Beckman Coulter), quantified with the Ion Library TaqMan Quantitation kit (Thermo Fisher Scientific), diluted to 75 pM, and pooled equally. Emulsion PCR, enrichment, and loading were performed on an Ion Chef Instrument. Templated libraries were sequenced on an Ion Proton system using the Ion P1 Hi-Q Chef kit and the Ion P1 Chip kit v.3 (Thermo Fisher Scientific). Ion Proton reads were analyzed using the AmpliSeqRNA analysis plugin (v.5.2.1.2) in Torrent Suite software (Thermo Fisher Scientific). The data are publically available via the NCBI GEO database (GSE110180).
6
Transcriptome Sequencing Protocol for Gene Expression
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For each sample, 50 ng of total RNA was reverse transcribed using the Ion AmpliSeq Transcriptome Human Gene Expression kit following the protocol of the manufacturer (Thermofisher Scientific®). The cDNA libraries were amplified and barcoded using Ion AmpliSeq Transcriptome Human Gene Expression core panel and Ion Xpress Barcode Adapter (Thermofisher Scientific®). The amplicons were quantified using Agilent High Sensitivity DNA kit before the samples were pooled in sets of eight. Emulsion PCR and enrichment was performed on the Ion OT2 system instrument using the Ion PI Hi-Q OT2 200 kit (Thermofisher Scientific®). Samples were loaded on an Ion PI v3 Chip and sequenced on the Ion Proton System using Ion PI Hi-Q sequencing 200 kit chemistry (200 bp read length; Thermofisher Scientific®). The quality control of the sequencing data was evaluated as described previously (57 (link)). The DEG between conditions was calculated with (v1.18.1 using R v3.4.1). Genes were considered differentially expressed when their adjusted P-value was <0.05 and their|log2FoldChange|was >0.4 (options: lfcThreshold = 0.4, altHypothesis = ‘greaterAbs’). Data are available on GEO database (GSE161029).
7
RNA-Seq Transcriptome Analysis using Ion AmpliSeq
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Total RNA was purified using an RNeasy Micro Kit (Qiagen) and treated with a DNase I kit (Qiagen) to remove genomic DNA. We reverse-transcribed 10 ng of total RNA to obtain single-stranded cDNA using the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific). We synthesized cDNA libraries for the Ion Ampliseq transcriptome analysis using the Ion AmpliSeq Transcriptome Human Gene Expression Core Panel (Thermo Fisher Scientific) and Ion Ampliseq Library Kit Plus (Thermo Fisher Scientific), according to the manufacturer’s protocol. Briefly, cDNA was amplified for 12 cycles with an Ion AmpliSeqTM Transcriptome Human Gene Expression Core Panel using a thermal cycler. Primer sequences were partially digested with the FuPa reagent by sequentially performing 10 min at 50°C, 10 min at 55°C, and 20 min at 60°C. Barcode ligation was performed using an Ion Xpress Barcode for 30 min at 22°C. Barcode-labeled cDNA libraries were purified using DNA Clean & Concentrator™-5 (Zymo Research, CA, United States) and analyzed using the Ion S5 XL System (Thermo Fisher Scientific) and Ion 540 Chip Kit (Thermo Fisher Scientific). Count data analyses were performed using R studio with the “DESeq2” package normalization method for the detection of significantly (p ≤ 0.05) differentially expressed genes (Love et al., 2014 (link)).
8
Transcriptome Profiling using Ion Proton
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For each sample, 50 ng of total RNA was reverse transcribed using the Ion AmpliSeq Transcriptome Human Gene Expression kit following the protocol of the manufacturer (Thermofisher Scientific®). Briefly, the cDNA libraries were amplified and barcoded using Ion AmpliSeq Transcriptome Human Gene Expression core panel and Ion Xpress Barcode Adapter, named Amplicons (Thermofisher Scientific®). The amplicons were quantified using Agilent High Sensitivity DNA kit before the samples were pooled in sets of eight. Emulsion PCR and Enrichment was performed on the Ion OT2 system Instrument using the Ion PI Hi-Q OT2 200 kit (Thermofisher Scientific). Samples were loaded on an Ion PI v3 Chip and sequenced on the Ion Proton System using Ion PI Hi-Q sequencing 200 kit chemistry to generated around 10.156.774 reads per sample (200 bp read length; Thermofisher Scientific). The fastQ file was generated on the Ion Torrent server and transfer to the PSMN (for scientific pole for numerical modeling at ENS Lyon; www.ens-lyon.fr/PSMN ).
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