ion xpress rna seq barcode 1 16 kit  (Thermo Fisher)

Journal: Journal of Extracellular Vesicles

Article Title: Dynamic release of neuronal extracellular vesicles containing miR‐21a‐5p is induced by hypoxia

doi: 10.1002/jev2.12297

Figure Lengend Snippet: Ischemic stroke alters the levels of miR‐21a‐5p in the brain and oxygen‐glucose deprived neurons. A . Heatmap of differentially expressed miRNAs from small RNA sequencing of the mouse brain tissue 3 days post‐permanent middle cerebral artery occlusion (pMCAO). B . Validation of miR‐21a‐5p alteration in the mouse brain tissue up to 5 days post‐pMCAO by qPCR (n = 5–6). One‐way ANOVA and Tukey's post hoc for each timepoint was performed. C . The regulation of miR‐21a‐5p levels were additionally studied in different primary and immortalised mouse cell lines in normoxia and oxygen‐glucose deprivation (OGD) at the 12‐h timepoint. The dots correspond to technical replicates (n = 4–6) and T‐test between treatment and control of each cell line was performed. D . Volcano plot showing changes in the transcriptional activity of miRNA genes in mouse primary cortical neurons after 6‐h glutamate exposure as quantified from GRO‐seq. Bar plot shows the quantification of fold change for miR‐21 (n = 2). BioConductor limma package ( Ritchie et al., 2015 ) that computes moderate t‐statistics and false discovery rates (Benjamini‐Hochberg) was performed. E . Pathway enrichment analysis of HITS‐clip miR‐21a‐5p target genes using Metascape. F,H . Network construction of enriched pathways using IPA. The list of miR‐21a‐5p bound putative targets in N2A cells were obtained from HITS‐clip analysis and extended with functionally connected genes using IPA software. G . Relative coverage and miRNA enrichment of miRNAs of interest obtained from HITS‐clip analysis. B, C and D data are represented as mean ± SD and the significance is stated as *** p < 0.001, ** p < 0.01, * p < 0.05

Article Snippet: The Ion Total RNA‐Seq Kit V2 (Life Technologies, Australia) was used to create small RNA libraries which were ligated to adapters containing a unique index barcode (Ion Xpress™ RNA‐Seq Barcode 1–16 Kit, Life Technologies, Australia) according to the manufacturers’ protocol.

Techniques: RNA Sequencing Assay, Activity Assay, Software

Journal: RNA Biology

Article Title: Defining the purity of exosomes required for diagnostic profiling of small RNA suitable for biomarker discovery

doi: 10.1080/15476286.2016.1270005

Figure Lengend Snippet: Workflow outlining the exosomal small RNA sequencing experiment and data analysis. (A) Exosomes released from GT1–7 neuronal cells found in the conditioned media were isolated using 2 different centrifugation-based protocols. Protocol 1 involves the use of differential UC and Protocol 2 involves isolating exosomes using differential UC over a continuous 10–30% Optiprep gradient. Upon exosome isolation small RNA was extracted from both cells and exosomes using TRIzol-LS followed by clean up using the column-based miRNeasy kit. The quality and quantity of the small RNA were checked by Agilent 2100 Bioanalyser. Libraries were constructed and subjected to small RNA transcriptome sequencing using Ion Torrent Personal Genome Machine. The initial bioinformatics analysis of pre-processing, quality assessment and sequence mapping (i.e. TMAP) were performed in Ion Torrent Suite. The generated high quality and aligned data was subjected to an in-house developed small RNA analysis pipeline that integrates BEDtools and edgeR for small RNA profiling. The pipeline automatically generates output of raw counts, normalized counts and vizualization graphics including, but not limited to, hierarchical clustering heatmap and multidimensional scaling. (B) Flowchart describing the steps of the 2 protocols used to isolate exosomes from the conditioned medium; (i) differential UC and (ii) Optiprep velocity gradient ultracentrifugation.

Article Snippet: For each individual library, RNA was ligated to adapters containing a unique index barcode (Ion Xpress™ RNA-Seq Barcode 1–16 Kit, Life Technologies) to allow libraries to be pooled during Ion Torrent sequencing (Life Technologies).

Techniques: RNA Sequencing Assay, Isolation, Centrifugation, Construct, Sequencing, Generated

Journal: RNA Biology

Article Title: Defining the purity of exosomes required for diagnostic profiling of small RNA suitable for biomarker discovery

doi: 10.1080/15476286.2016.1270005

Figure Lengend Snippet: Read length of small RNA sequencing. Read length (nucleotide) distribution after adaptor removal. Each y-axis depicts the number of sequencing reads of all samples comprising of 5 replicates in each of the respective fraction, 3 replicates of exosomes isolated from ultracentrifugation method, and 3 replicates of GT1–7 neuronal cells. Standard deviations are shown in the bar graph.

Article Snippet: For each individual library, RNA was ligated to adapters containing a unique index barcode (Ion Xpress™ RNA-Seq Barcode 1–16 Kit, Life Technologies) to allow libraries to be pooled during Ion Torrent sequencing (Life Technologies).

Techniques: RNA Sequencing Assay, Sequencing, Isolation

Journal: RNA Biology

Article Title: Defining the purity of exosomes required for diagnostic profiling of small RNA suitable for biomarker discovery

doi: 10.1080/15476286.2016.1270005

Figure Lengend Snippet: Small RNA sequencing of GT1–7 neuronal cells and GT1–7-derived exosomes isolated by 2 different protocols. (A) Circos diagram depicting small RNA transcriptome data. Track A: cytogenetic bands, chromosomes are depicted qter to pter. Track B to G: Highlight of all small RNAs (miRNA, rRNA, tRNA, piRNA, snRNA and snoRNA) identified across different sample groups in respect to GT1–7 neuronal cell, exosomes isolated from differential UC (protocol 1) and Optiprep fractions (i.e. Fraction 7 to 10) using Optiprep velocity gradient ultracentrifugation (protocol 2). (B) Distribution of small RNA species in the analyzed data sets. Profiles of different biotypes in each data set are percentage proportion to the total reads mapped to small RNA species and other elements, in particular to tRNA, rRNA, snoRNA, snRNA, piwi-RNA, miRNA, protein-coding fragments and others (i.e. repeat elements). Abbreviations: ‘miRNA’ – microRNA; ‘rRNA’ – rRNA; ‘tRNA’ – tRNA; ‘piRNA’ – piwi-interacting RNA; ‘snRNA’ – small nuclear RNA; ‘snoRNA’ – small nucleolar RNA; ‘UCexo’ – exosomes from differential UC.

Article Snippet: For each individual library, RNA was ligated to adapters containing a unique index barcode (Ion Xpress™ RNA-Seq Barcode 1–16 Kit, Life Technologies) to allow libraries to be pooled during Ion Torrent sequencing (Life Technologies).

Techniques: RNA Sequencing Assay, Derivative Assay, Isolation