RNA quality was assessed by the absence of smear of 18S and 28S bands as analyzed by the Bio analyzer 2100 (Agilent Technologies, Santa Clara, CA). For the construction of micro RNA libraries, IonTotal RNA-Seq Kit v2 and Ion Xpress™ RNA-Seq Barcode kit (Life Technologies, Foster City, CA) were used. The Ion Total RNA-Seq Kit v2 (Life Technologies) was used for the preparation of micro RNA libraries according to the manufacturer’s instructions starting with 100 ng total RNA. The template was prepared from the libraries using the Ion OneTouch™ System (Life Technologies) and sequenced on an Ion PGM™ Sequencer (Life Technologies). Total read counts are shown in Additional file 5 : Table S5. As a preprocessing step, the sequencing adapter was trimmed and read to be over the range of miRNA length (18–30 nucleotides) were excluded. We allowed bowtie2 with options of one mismatch and used mouse miRbase ver. 19 as a reference. [42 (link), 43 (link)]. After alignment, a total of 527 miRNAs were identified. Raw level data from the miRNA read count were normalized using the quintile method and considered as miRNA expression. Differentially expressed miRNA (DEmiR) was also tested using limma-like microarray conditions. Finally 54 DEmiRs were detected within a cutoff p-value ≤ 0.1.
Ion total rna seq kit v2
Manufactured by Thermo Fisher Scientific
Sourced in United States, Australia, Japan, Netherlands
The Ion Total RNA-Seq Kit v2 is designed for library preparation and sequencing of total RNA samples using the Ion Torrent sequencing platform. The kit enables the preparation of cDNA libraries from total RNA for subsequent sequencing on the Ion Torrent system.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (38 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (27 mentions)
Ion proton sequencer, by Thermo Fisher Scientific (26 mentions)
2100 bioanalyzer, by Agilent Technologies (23 mentions)
Ion proton system, by Thermo Fisher Scientific (21 mentions)
Rneasy mini kit, by Qiagen (19 mentions)
Dynabeads mrna direct micro kit, by Thermo Fisher Scientific (18 mentions)
Ion xpress rna seq barcode 1 16 kit, by Thermo Fisher Scientific (15 mentions)
Ion pi hi q chef kit, by Thermo Fisher Scientific (15 mentions)
Ion pi chip, by Thermo Fisher Scientific (13 mentions)
Dynabeads mrna direct purification kit, by Thermo Fisher Scientific (12 mentions)
Ion pi hi q sequencing 200 kit, by Thermo Fisher Scientific (11 mentions)
Dynabeads mrna direct micro purification kit, by Thermo Fisher Scientific (11 mentions)
Ion pi sequencing 200 kit v2, by Thermo Fisher Scientific (11 mentions)
Ion pi template ot2 200 kit v3, by Thermo Fisher Scientific (10 mentions)
Ion 540 chip, by Thermo Fisher Scientific (9 mentions)
Ion onetouch system, by Thermo Fisher Scientific (9 mentions)
Onetouch 2 instrument, by Thermo Fisher Scientific (9 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (9 mentions)
Ion pi hi q ot2 200 kit, by Thermo Fisher Scientific (9 mentions)
280 protocols using ion total rna seq kit v2
1
Profiling Murine miRNA Transcriptome
2
RNA-Seq library preparation and sequencing
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Total RNA pools from each sample were then used for preparing cDNA libraries using the Ion Total RNA‐Seq kit V2 (Life Technologies Corporation, CA, USA). Double‐stranded cDNA was ligated to barcoded adapters and was sequenced using the Ion PI™ Chip (Ion torrent, Life technologies, CA, USA) at the Beijing Genomics Institute (Shenzhen, China). Libraries were run at a concentration of 4–5 pM. To ensure the accuracy of subsequent analysis, raw sequences were cleaned to remove adaptors and sequencing errors. Reads were removed if they contained the sequencing adaptor, more than 5% unknown nucleotides, or more than 20% of bases of low quality. This output was called clean reads, which was used for subsequent downstream analyses.
3
Ion RNA-Seq Library Preparation
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Library preparation was performed using the Ion Total RNA-Seq Kit v2 (Life Technologies). Large RNA libraries were prepared using the whole transcriptome protocol provided with the kit, and small RNA libraries were prepared using the small RNA protocol. Large and small RNA libraries were barcoded using Ion Xpress Barcode Adapters (Life Technologies). Quality control was performed using Agilent High Sensitivity chips (Agilent) and Experion DNA 1K kits (Bio-Rad). Libraries were loaded onto Ion PI chips via the Ion PI Template OT2 200 v3 (for small RNAs) or v2 (for large RNAs), and Ion PI Sequencing 200 v3 (for small RNAs) or v2 (for large RNAs) Kits, and sequenced on an Ion Proton Sequencer (Life Technologies).
4
Bacterial Transcriptome Profiling from Infected Lungs
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In vitro and infected mouse lung tissue samples were thawed and nucleic acid was isolated by organic partition. Samples were treated with DNAse (Fermentas, Burlington, Ontario) for 30 minutes and purified by phenol/chloroform/isoamyl alcohol (25:24:1) (Fisher Scientific, Pittsburgh, PA) extraction and ammonium acetate precipitation. Biological replicates (not pooled) were submitted to the CSU Next Generation Sequencing core for sample processing and sequencing. Briefly, RNA sample quality was determined on an Agilent 2100 Bioanylizer and samples with a RIN value greater than 8 passed the criteria for sequencing. Host transcripts were removed using MICROBEnrich (Life Technologies, Carlsbad, CA), sample libraries were prepared using the Ion Total RNA-Seq kit v2 (Life Technologies), and multiplexed on a P1 chip using Ionxpress RNA-Seq 1–16 kit (Life Technologies). Whole bacterial transcriptome sequencing was performed using the Ion Proton Next Generation Sequencer (Life Technologies).
5
RNA-seq Analysis of Mouse Transcriptome
6
Ion Total RNA-Seq Library Preparation
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Method 1 for the library preparation was based on the Ion Total RNA-Seq kit v2 (Life Technologies, USA) according to the manufacturer’s instructions. Briefly, the extracted viral RNA was quantified, and then digested using RNase III. The fragmented RNA was ligated with random adaptors, and this was followed by reverse transcription. The cDNA was amplified through PCR, and the amplicons around 450 bp were collected with the E-Gel® SizeSelect™ Agarose Gel (Life Technologies, USA).
7
Total RNA Extraction and RNA-seq
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Total RNA was extracted using TRIzol reagent (Invitrogen Carlsbad, CA, USA) and treated with RNase free DNase I (Qiagen, Hilden, Germany) to remove any DNA contamination. Total RNA thus obtained was subjected to RNAseq library preparation as per the Ion total RNAseq kit v2 (Life technologies, Carlsbad, CA, USA). The sequencing was carried on Ion torrent PGM using 316 chip (Life technologies, Carlsbad, CA, USA).
8
Colon Tissue RNA-seq Analysis Pipeline
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Three group samples (control, DSS, and MXSP groups) were selected for RNA-seq analysis. Three biological repeats were performed for each group. TRIzol reagent (Invitrogen, United States) was used to extract total mRNA from the colon tissue according to the standard protocol. RNA quantity and purity were detected by using a Nanodrop 2000. The RNA integrity and renewable identification numbers (RIN) were verified using an Agilent 2100 Bioanalyzer. The mRNA sequencing library was constructed using an Ion Total RNA-Seq Kit v2 (Life Technologies, United States), according to the manufacturer’s instructions. Then, the cDNA libraries were sequenced using an Illumina HiSeq 2000. The following de novo assembly and bioinformatic analysis were performed on the Majorbio cloud platform (Majorbio, Shanghai, China). Differential expression genes (DEGs) of samples were determined using DEGseq (http://bioconductor.org/packages/stats/bioc/DEGSeq/ ), DESeq2 (http://bioconductor.org/packages/stats/bioc/DESeq2/ ), and edgeR (http://bioconductor.org/packages/stats/bioc/edgeR/ ).
9
Profiling RNA Expression in Abdominal Aortic Aneurysm
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The procedure of study material preparation and sequencing was conducted as previously described in [28 (link)].
Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood specimens using density gradient centrifugation with Gradisol L reagent (Aqua-Med, Łódź, Poland). Proportions of white blood cells subpopulations in AAA group were obtained from venous blood morphology analysis results and were presented inFigure S1 .
Small RNA fractions (for miRNA expression analysis) were isolated from PBMCs specimens of twenty eight AAA patients and nineteen control subjects using MirVana microRNA Isolation Kit (Ambion, Austin, TX, USA).
Total RNA specimens (for transcriptome analysis) were isolated from PBMCs samples of seven randomly selected AAA patients and seven randomly selected controls using TRI Reagent Solution (Applied Biosystems, Foster City, CA, USA).
Small RNA and transcriptome libraries were prepared using Ion Total RNA-Seq Kit v2, Magnetic Bead Cleanup Module kit, Ion Xpress RNA-Seq Barcode 01-16 Kit and sequenced on Ion 540 chips (all Life Technologies, Carlsbad, CA, USA) using Ion S5 XL System (Thermo Fisher Scientific, Waltham, MA, USA). Raw sequences of small RNA and transcriptomic libraries were aligned to 2792 human miRNAs from miRBase v21 (http://www.mirbase.org ) and to 55,765 genes of hg19 human genome, respectively.
Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood specimens using density gradient centrifugation with Gradisol L reagent (Aqua-Med, Łódź, Poland). Proportions of white blood cells subpopulations in AAA group were obtained from venous blood morphology analysis results and were presented in
Small RNA fractions (for miRNA expression analysis) were isolated from PBMCs specimens of twenty eight AAA patients and nineteen control subjects using MirVana microRNA Isolation Kit (Ambion, Austin, TX, USA).
Total RNA specimens (for transcriptome analysis) were isolated from PBMCs samples of seven randomly selected AAA patients and seven randomly selected controls using TRI Reagent Solution (Applied Biosystems, Foster City, CA, USA).
Small RNA and transcriptome libraries were prepared using Ion Total RNA-Seq Kit v2, Magnetic Bead Cleanup Module kit, Ion Xpress RNA-Seq Barcode 01-16 Kit and sequenced on Ion 540 chips (all Life Technologies, Carlsbad, CA, USA) using Ion S5 XL System (Thermo Fisher Scientific, Waltham, MA, USA). Raw sequences of small RNA and transcriptomic libraries were aligned to 2792 human miRNAs from miRBase v21 (
10
Exosomal RNA Sequencing Protocol
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PAXgene, cell-free and exosomal RNA was converted into cDNA libraries using the Ion Total RNA-Seq Kit V2 (Life Technologies, Australia) and prepared for deep sequencing as described previously (12 (link)). Pooled libraries with unique barcodes were loaded on 318 sequencing chips and run on the Ion Torrent Personal Genome Machine (Life Technologies, Australia). The Torrent Suite 3.4.1 was used to manage the Ion Torrent PGM to process raw signals and perform base calling. The sequences are then assessed for quality and primer-adapter sequences are trimmed by the Torrent Suite software, followed by alignment to the human reference genome (HG19). The trimmed and aligned data was transferred to Partek Genomics Suite and mapped to known miRNA using miRBase V.20 and Ensembl Release 74. The number of reads for each miRNA were normalized to reads per million (RPM) across all samples. Samples containing less than 5 RPM were removed. Partek Genomics suite and statistical package was used to perform statistical analysis, hierarchical clustering and to identify unique miRNA in each sample type. Data have been uploaded to Vesiclepedia (http://microvesicles.org/ ).
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