Total RNA was isolated from the livers of mice fed the 16-week diets using a QIAGEN RNeasy Mini Kit (QIAGEN GmbH, Hilden, Germany). Ribosomal RNA was depleted using the RiboMinus Eukaryote System (Life Technologies, Carlsbad, CA, USA), and the remaining RNA was purified, fragmented, barcoded and reverse-transcribed using the Ion Total RNA-Seq Kit (Life Technologies). Sequencing was performed using Ion Torrent technology (Life Technologies), and whole-RNA expression analysis was performed using the Genomics Workbench system (CLC bio, Aarhus, Denmark). The expression of each gene was quantified as reads per kilobase of exon model per million mapped reads (RPKM). The average RPKM was calculated for the regular diet and high-fat diet groups, and their fold changes and P values were evaluated using Student’s t test. An absolute fold change of >1.5 and a P value > 0.05 were used to identify altered gene expression. Altered genes were further subjected to pathway analysis using MetaCore (GeneGo/Thomson Reuters, New York, NY, USA).
Ion total rna seq kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Ion Total RNA-Seq Kit is a laboratory equipment product designed for RNA sequencing. It provides a streamlined workflow for the preparation and sequencing of total RNA samples. The kit includes reagents and consumables necessary for the library preparation and sequencing process.
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Lab products found in correlation
Ion pi template ot2 200 kit, by Thermo Fisher Scientific (4 mentions)
Ion proton sequencer, by Thermo Fisher Scientific (4 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Ion pi sequencing 200 kit, by Thermo Fisher Scientific (3 mentions)
Ion pi chip kit, by Thermo Fisher Scientific (3 mentions)
Ion proton system, by Thermo Fisher Scientific (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Trizol reagent kit, by Thermo Fisher Scientific (2 mentions)
Microbenrich, by Thermo Fisher Scientific (2 mentions)
Agilent bioanalyzer hs dna kit, by Agilent Technologies (2 mentions)
Megaclear, by Thermo Fisher Scientific (2 mentions)
Microbexpress, by Thermo Fisher Scientific (2 mentions)
Ampure bead purification, by Beckman Coulter (2 mentions)
Mirvana kit, by Thermo Fisher Scientific (2 mentions)
Lysing matrix b, by MP Biomedicals (2 mentions)
Dynabeads mrna direct kit, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Ion pi chip v3, by Thermo Fisher Scientific (2 mentions)
Torrent suite software, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
23 protocols using ion total rna seq kit
1
Liver Transcriptome Profiling of Mice on Diets
2
miRNA Sequencing Library Preparation
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The amount of miRNA used in the following steps was targeted at 1–25 ng, maintaining the total RNA amount to less than 1 μg in a volume of 3 μL. The libraries were prepared with the Ion Total RNa-seq Kit (Life Technologies, Carlsbad, CA, USA), according to manufacturer’s instructions, and the final amplified product size was about 105 bp. The sequencing reaction was performed on an Ion Proton platform (Life Technologies, Carlsbad, CA, USA) setting 160 flows and the mean fragment size was about 20–25 bp.
3
Bacterial Transcriptome RNA Sequencing
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Aliquots of 2 ml were centrifuged for sediment the cells, at 5000 g
and 4°C for 10 minutes. The bacterial total RNA was extracted using
ChargeSwitch® Total RNA Cell Kits(Invitrogen, USA) according to the manufacturer’s instructions, and
adding a mechanical lysis step using a Precellys© grinder (Bertin Technologies, France). The total RNA
amount was measured by fluorometry using a Qubit™
- Quant-iT™ RNA Assay Kit (Invitrogen, USA). Messenger RNA
was enriched using Ribominus™ Transcriptome
Isolation Kit (Yeast and
Bacteria) (Invitrogen, USA), and the amount of recovered
RNA was measured as described above.
The enriched RNA samples were used to build strand-specific RNA-Seq
libraries. Two libraries, one at each experimental condition (0°C and
37°C, designated R1-0 and R1-37, respectively), were prepared using a
SOLiD™ Total RNA-Seq Kit. Fifty-base pair (bp) fragments were sequenced
using the SOLiD™ 3 Plus system according to the manufacturer’s
instructions (Applied Biosystems, USA). Two replicates from each
experimental condition (0°C and 37°C, designated as R2-0 and R3-0 and as
R2-37 and R3-37, respectively) were employed in the preparation of
fragment libraries using Ion Total RNA-Seq Kit and sequenced using 316
chip in an Ion Torrent Personal Genome Machine™ (Life Technologies, USA).
All the procedures were performed according to the manufacturer’s
instructions.
and 4°C for 10 minutes. The bacterial total RNA was extracted using
ChargeSwitch® Total RNA Cell Kits(Invitrogen, USA) according to the manufacturer’s instructions, and
adding a mechanical lysis step using a Precellys© grinder (Bertin Technologies, France). The total RNA
amount was measured by fluorometry using a Qubit™
- Quant-iT™ RNA Assay Kit (Invitrogen, USA). Messenger RNA
was enriched using Ribominus™ Transcriptome
Isolation Kit (Yeast and
Bacteria) (Invitrogen, USA), and the amount of recovered
RNA was measured as described above.
The enriched RNA samples were used to build strand-specific RNA-Seq
libraries. Two libraries, one at each experimental condition (0°C and
37°C, designated R1-0 and R1-37, respectively), were prepared using a
SOLiD™ Total RNA-Seq Kit. Fifty-base pair (bp) fragments were sequenced
using the SOLiD™ 3 Plus system according to the manufacturer’s
instructions (Applied Biosystems, USA). Two replicates from each
experimental condition (0°C and 37°C, designated as R2-0 and R3-0 and as
R2-37 and R3-37, respectively) were employed in the preparation of
fragment libraries using Ion Total RNA-Seq Kit and sequenced using 316
chip in an Ion Torrent Personal Genome Machine™ (Life Technologies, USA).
All the procedures were performed according to the manufacturer’s
instructions.
4
Whole Transcriptome Sequencing of Liver Cell Lines
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For whole transcriptome sequencing, RNA from 1×107 SMMC-7721 and HL-7702 cells was extracted using the TRIzol reagent kit (Invitrogen, Waltham, MA, USA) and then quantified using NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). The whole transcriptome RNA-seq procedure was performed using the Ion Total RNA-Seq kit, the Ion PI™ Chip kit, the Ion PI™ Template OT2 200 kit, and the Ion PI™ Sequencing 200 kit based on the protocols of Life Technologies Corp. (Waltham, MA, USA). In brief, mRNA was purified using oligo-dT beads from 100 µg of total RNAs for each sample and then fragmented. The cleaved RNA fragments were reverse-transcribed into First-Strand cDNA, followed by Second-Strand cDNA synthesis. Then, a single 'A' base was added to the cDNA fragments at the 3′ end. The cDNAs were ligated to adapters and enriched by polymerase chain reaction (PCR) to generate the final cDNA library. After amplifying the sequencing template, RNA-seq was performed using the Ion Proton System (Life Technologies) with the standard protocol.
5
Whole Transcriptome Sequencing of L02 and SMMC7721 Cells
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For whole transcriptome sequencing, RNA from 1×107 L02 and SMMC7721 cells were extracted using Trizol (Invitrogen, MA, USA) and then quantified by NanoDrop 2000 (Thermo-Fisher Scientific, MA, USA). The whole transcriptome RNA-seq was performed using the Ion Total RNA-Seq Kit, Ion PI™ Chip kit, Ion PI™ Template OT2 200 Kit, and Ion PI™ Sequencing 200 Kit based on the Life Technologies Corporation’s guide. In brief, mRNA was purified using oligo-dT beads from 100 μg of total RNAs for each sample and then fragmented. The cleaved RNA fragments were reverse-transcribed into first-strand cDNA, followed by second-strand cDNA synthesis. Then a single ‘A’ base was added to cDNA fragments at the 3′ end. The cDNAs were ligated to adapters, enriched by polymerase chain reaction (PCR) to generate the final cDNA library. After amplifying the sequencing template, RNA-seq was performed using the Ion proton system (Life Technologies Corporation, MA, USA) with the standard protocol.
6
Viral Genome Sequencing from Cellular Samples
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RNA was extracted from either 100 to 200 µL larvae homogenates (RR200) or cell culture supernatants (QQ40 1st passage and QQ40 10th passage; see Table 1 ) using, respectively, a NucleoSpin virus kit (Macherey-Nagel, Hoerdt, France) or an Adiamag kit (Bio-X Diagnostics, Belgium) coupled with a KingFisher Duo Prime instrument (ThermoFisher Scientific Inc., Worcester, MA, USA) following the manufacturer’s instructions. cDNA libraries were prepared with an Ion Total RNA-Seq Kit (Life Technologies, Carlsbad, CA, USA) following supplier’s instructions. The cDNA libraries were sequenced using an Ion Proton Sequencer and Ion PI Chip v3 (Life Technologies, Carlsbad, CA, USA). The reads were first mapped onto the host’s genome using Bowtie 2 [27 (link)]. Unmapped reads were then assembled with SPades (v3.10.0, option-careful) [28 (link)] and the de novo contigs were BLASTed against the NCBI nr nucleotide or protein databases for identification of viral sequences.
7
MRSA RNA Sequencing Protocol
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RNA from both the ex vivo and in vitro samples were processed identically from all subjects and MRSA isolates. Total RNA was extracted using the Mirvana kit (Life Technologies, Grand Island, NY), with the inclusion of a bead-beating step for 20 min with Lysing-Matrix B (MP Bio, Santa Ana, CA). Total RNA then was enriched for microbial RNA using MicrobEnrich (Life Technologies), and further enriched for mRNA using MicrobExpress (Life Technologies) and MegaClear (Life Technologies), which are designed to remove ribosomal RNAs. Enriched RNA then was prepared for sequencing through the construction of cDNA libraries using the Ion Total RNA-Seq kit (Life Technologies), and subjected to successive rounds of Ampure bead purification (Beckman-Coulter, Brea, CA) to remove small cDNAs. Libraries were quantified using an Agilent Bioanalyzer HS DNA Kit (Agilent, Santa Clara, CA) and then were sequenced on a 314 chips using an Ion Torrent Personal Genome Machine (Rothberg et al., 2011 (link)), producing an average of 559,129 reads per subject of mean length 106 nucleotides. All sequence data produced in this study are available in the MG-Rast database (metagenomics.anl.gov/) under the project name “MRSA_RNAseq_Study” or project #2278.
8
RNA Sequencing for CSpV1 Genome Assembly
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CSpV1 genome was obtained from a pool of samples B44 and B45, using RNA sequencing. cDNA libraries were prepared using an Ion total RNAseq Kit (Life technologies, Carlsbad, CA, USA) according to the supplier’s instructions. The cDNA libraries were sequenced using an Ion Proton Sequencer and an Ion PI Chip v2 (Life technologies).
Sequence reads were cleaned and trimmed for adapter removal using fastP version 0.20.1 [34 (link)] and sequence quality was verified using FastQC version 0.11.8 [35 ]. Reads were assembled with rnaSPADES de novo assembler as implemented in SPAdes assembler version 3.10.0 [36 (link)]. Resulting contigs were aligned on local nt database with Megablast version 2.10.1 to identify viral references. Then, both sequence reads and assembled contigs were aligned using Burroughs-Wheeler Aligner (BWA, version 0.7.8) [37 (link)] against the CSpV1 strain Iowa genome fragments (NC_038843 dsRNA1 and NC_038844 dsRNA2) and visualized in Integrative Genome Viewer (IGV) [38 (link), 39 (link)] to control the quality of the consensus sequence extracted using Samtools pileup version 1.8 [40 (link)].
Sequence reads were cleaned and trimmed for adapter removal using fastP version 0.20.1 [34 (link)] and sequence quality was verified using FastQC version 0.11.8 [35 ]. Reads were assembled with rnaSPADES de novo assembler as implemented in SPAdes assembler version 3.10.0 [36 (link)]. Resulting contigs were aligned on local nt database with Megablast version 2.10.1 to identify viral references. Then, both sequence reads and assembled contigs were aligned using Burroughs-Wheeler Aligner (BWA, version 0.7.8) [37 (link)] against the CSpV1 strain Iowa genome fragments (NC_038843 dsRNA1 and NC_038844 dsRNA2) and visualized in Integrative Genome Viewer (IGV) [38 (link), 39 (link)] to control the quality of the consensus sequence extracted using Samtools pileup version 1.8 [40 (link)].
9
RNA-Seq Library Preparation Protocol
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For library construction, we followed the protocol of the Ion Total RNA-Seq Kit
(Life Technologies, Carlsbad, California, USA). Ion OneTouch 200 Template Kit v2
DL was used for emulsion PCR and sequencing was performed with Ion PGM 200
Sequencing Kit (180 flows).
(Life Technologies, Carlsbad, California, USA). Ion OneTouch 200 Template Kit v2
DL was used for emulsion PCR and sequencing was performed with Ion PGM 200
Sequencing Kit (180 flows).
10
Transcriptome Profiling of VEEV-Infected U87MG Cells
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RNA from U87MG cells was purified from both VEEV TrD-infected (biosafety level 3) and mock-infected U87MG cells at 4, 8, and 16 hpi utilizing a mirVana isolation kit (Life Technologies). Quality control of purified RNA was then performed using an Agilent 2100 bioanalyzer, and an RNA integrity number (RIN) cutoff of 8 was utilized for all samples. An External RNA Controls Consortium (ERCC) RNA spike-in control mix was then added to the total RNA inputs (10 μg RNA) before poly(A) selection using a Life Technologies Dynabeads mRNA Direct kit. Preparation of a whole-transcriptome RNA library from purified mRNA was then performed using an Ion Total RNA-Seq kit (v2; Life Technologies). Quality control of the cDNA libraries was then performed using the Agilent 2100 bioanalyzer along with sterility testing for removal of libraries for sequencing from a BSL-3 to BSL-2 laboratory.
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