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Comparison or mRNA-seq libraries from <t>oligo-dT</t> and Cap-captured mRNA. A-B. Reads from dT or Cap-capture prepared libraries were aligned to sequences consisting of the 5’ or 3’ 25% of Refseq <t>mRNAs.</t> Reads aligning to the 5’ and 3’ portions of the transcript are plotted. Blue line indicates a ratio of one. C. Reads from mRNA-seq libraries prepared using oligo-dT captured mRNAs from mitotic and interphase Xenopus egg extract were aligned to the Xenopus laevis Unigene database. Relative abundance of each mRNA in mitotic and interphase extracts is plotted. Red points highlight two-fold differences between mitotic and interphase samples. D. Same experiment as in panel C except that the mRNAs were purified using Cap-capture prior to library preparation. Red points highlight two-fold differences. E. Scatterplot of mRNA abundance in oligo-dT-captured and Cap-captured mRNA libraries. F. The ratio of reads per mRNA in mitotic and interphase extracts are plotted for oligo-dT captured mRNAs (X-axis, data from panel A) and cap-captured mRNAs (Y-axis data from panel B). The colored points correspond to mRNAs with known changes in poly-A tail length (cdk1, green; Eg2/aurora-a, orange; mos, red; Xlcl1, yellow). Quadrants highlight two-fold differences between samples.
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1) Product Images from "Combining Different mRNA Capture Methods to Analyze the Transcriptome: Analysis of the Xenopus laevis Transcriptome"

Article Title: Combining Different mRNA Capture Methods to Analyze the Transcriptome: Analysis of the Xenopus laevis Transcriptome

Journal: PLoS ONE

doi: 10.1371/journal.pone.0077700

Comparison or mRNA-seq libraries from oligo-dT and Cap-captured mRNA. A-B. Reads from dT or Cap-capture prepared libraries were aligned to sequences consisting of the 5’ or 3’ 25% of Refseq mRNAs. Reads aligning to the 5’ and 3’ portions of the transcript are plotted. Blue line indicates a ratio of one. C. Reads from mRNA-seq libraries prepared using oligo-dT captured mRNAs from mitotic and interphase Xenopus egg extract were aligned to the Xenopus laevis Unigene database. Relative abundance of each mRNA in mitotic and interphase extracts is plotted. Red points highlight two-fold differences between mitotic and interphase samples. D. Same experiment as in panel C except that the mRNAs were purified using Cap-capture prior to library preparation. Red points highlight two-fold differences. E. Scatterplot of mRNA abundance in oligo-dT-captured and Cap-captured mRNA libraries. F. The ratio of reads per mRNA in mitotic and interphase extracts are plotted for oligo-dT captured mRNAs (X-axis, data from panel A) and cap-captured mRNAs (Y-axis data from panel B). The colored points correspond to mRNAs with known changes in poly-A tail length (cdk1, green; Eg2/aurora-a, orange; mos, red; Xlcl1, yellow). Quadrants highlight two-fold differences between samples.
Figure Legend Snippet: Comparison or mRNA-seq libraries from oligo-dT and Cap-captured mRNA. A-B. Reads from dT or Cap-capture prepared libraries were aligned to sequences consisting of the 5’ or 3’ 25% of Refseq mRNAs. Reads aligning to the 5’ and 3’ portions of the transcript are plotted. Blue line indicates a ratio of one. C. Reads from mRNA-seq libraries prepared using oligo-dT captured mRNAs from mitotic and interphase Xenopus egg extract were aligned to the Xenopus laevis Unigene database. Relative abundance of each mRNA in mitotic and interphase extracts is plotted. Red points highlight two-fold differences between mitotic and interphase samples. D. Same experiment as in panel C except that the mRNAs were purified using Cap-capture prior to library preparation. Red points highlight two-fold differences. E. Scatterplot of mRNA abundance in oligo-dT-captured and Cap-captured mRNA libraries. F. The ratio of reads per mRNA in mitotic and interphase extracts are plotted for oligo-dT captured mRNAs (X-axis, data from panel A) and cap-captured mRNAs (Y-axis data from panel B). The colored points correspond to mRNAs with known changes in poly-A tail length (cdk1, green; Eg2/aurora-a, orange; mos, red; Xlcl1, yellow). Quadrants highlight two-fold differences between samples.

Techniques Used: Purification

Poly-A tail analysis of selected mRNAs. mRNAs that exhibited changes in Mitosis:Interphase (M:IF) abundance ratios in oligo-dT-captured samples, but not in Cap-captured samples were analyzed for poly-A tail length using the ePAT assay and anchored TVN reverse transcription controls. A. Six mRNAs with high M:IF ratios (aurora-a, esco2, fbox5, stx11, march7, and hexim1) showed longer poly-A tails in mitotic extract. Two mRNA (setd8 and MGC83922) with a low M:IF ratio showed very modest changes in poly-A tail lengths between Mitosis and Interphase. Red asterix indicates the position of the prominent TVN PCR product that is quantified in B. B. The amount of PCR product contained in the TVN-RT PCR reactions (red asterix in A) were quantified. The ratio of the amount of PCR product in Mitotic to Interphase extracts is presented in the first line. The ratio of each mRNA in Mitotic and Interphase extracts as determined by RNA-seq is presented below the PCR derived ratios for comparison. In addition five mRNAs with high M:IF ratios (aurora-1, fbox5, stx11, march7, and hexim1) had increased amounts of minimal poly-A tail PCR products in mitosis compared to interphase in TVN controls (quantified below gel) while both setd8 and MGC88922 had higher levels of TVN PCR products in interphase compared to mitotic extracts TVN PCR products indicate mRNAs with poly-A tails of 18 As. Line traces of ePAT PCR reactions presented in panel A. Black lines indicate traces from mitotic extract and red lines indicate traces from interaphse extract. D. Semi-quantitative PCR for each of the mRNAs tested in A was performed on RNA from mitotic and interphase extracts. Random hexamers were used to prime reverse transcription for these reactions. A second experiment showing very similar results is present in Figure S2.
Figure Legend Snippet: Poly-A tail analysis of selected mRNAs. mRNAs that exhibited changes in Mitosis:Interphase (M:IF) abundance ratios in oligo-dT-captured samples, but not in Cap-captured samples were analyzed for poly-A tail length using the ePAT assay and anchored TVN reverse transcription controls. A. Six mRNAs with high M:IF ratios (aurora-a, esco2, fbox5, stx11, march7, and hexim1) showed longer poly-A tails in mitotic extract. Two mRNA (setd8 and MGC83922) with a low M:IF ratio showed very modest changes in poly-A tail lengths between Mitosis and Interphase. Red asterix indicates the position of the prominent TVN PCR product that is quantified in B. B. The amount of PCR product contained in the TVN-RT PCR reactions (red asterix in A) were quantified. The ratio of the amount of PCR product in Mitotic to Interphase extracts is presented in the first line. The ratio of each mRNA in Mitotic and Interphase extracts as determined by RNA-seq is presented below the PCR derived ratios for comparison. In addition five mRNAs with high M:IF ratios (aurora-1, fbox5, stx11, march7, and hexim1) had increased amounts of minimal poly-A tail PCR products in mitosis compared to interphase in TVN controls (quantified below gel) while both setd8 and MGC88922 had higher levels of TVN PCR products in interphase compared to mitotic extracts TVN PCR products indicate mRNAs with poly-A tails of 18 As. Line traces of ePAT PCR reactions presented in panel A. Black lines indicate traces from mitotic extract and red lines indicate traces from interaphse extract. D. Semi-quantitative PCR for each of the mRNAs tested in A was performed on RNA from mitotic and interphase extracts. Random hexamers were used to prime reverse transcription for these reactions. A second experiment showing very similar results is present in Figure S2.

Techniques Used: Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, RNA Sequencing Assay, Derivative Assay, Real-time Polymerase Chain Reaction

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RNA Extraction:

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Labeling:

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Sequencing:

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Article Snippet: Paragraph title: mRNA-seq libraries preparation and deep sequencing ... RNA was extracted separately for each mouse by RNeasy Plus Mini Kit (Qiagen) and then pooled for WT or Taf7l KO samples; 4 μg of each RNA pool was used to purify mRNA using oligo (dT) and subsequently converted into multiplexed mRNA-seq libraries using mRNA-Seq Trueseq Kit (Illumina, San Diego, CA).

Article Title: Responses of the Housefly, Musca domestica, to the Hytrosavirus Replication: Impacts on Host's Vitellogenesis and Immunity
Article Snippet: RNA-Seq library construction and data analysis Preparation and sequencing of RNA libraries were performed by ICBR/UF (Gainesville, FL) according to the manufacturer's instructions (Illumina, Inc., San Diego, CA) using the NextSeq500 platform. .. Briefly, the mRNA was enriched from 1 μg of total RNA per sample using oligo-dT attached to magnetic beads and then subjected to thermal fragmentation using the elute, prime, and fragmentation mix from the Illumina TruSeq™ v2 RNA sample preparation kit.

Article Title: Transcriptomic profiling of the salt-stress response in the wild recretohalophyte Reaumuria trigyna
Article Snippet: .. cDNA library construction and sequencing Beads with Oligo (dT) (Illumina) were used to isolate poly (A) + RNA from 20 μg of each RNA pool. .. Fragmentation buffer (Ambion, Austin, TX, USA) was added to break mRNA into short fragments.

Article Title: Functional characterization of three trehalase genes regulating the chitin metabolism pathway in rice brown planthopper using RNA interference
Article Snippet: Paragraph title: cDNA library construction and high-throughput sequencing ... The fragments apart from the 3′ cDNA fragments connected to Oligo (dT) were washed away, and the Illumina adaptor 1 was ligated to the 5′ cohesive ends.

Article Title: Spatially resolved transcriptomics reveals plant host responses to pathogens
Article Snippet: Data quality control and mapping The sequencing reads were quality controlled using FastQC-0.11.5 [ ]. .. After quality control we used cutadapt-1.17 [ ] to trim low-quality bases (-q20) and remove Oligo-dT, template switching oligos, primer and Illumina Nextera library preparation sequences (−n 5 −e 0.05 −overlap 10).

cDNA Library Assay:

Article Title: De novo transcriptome assembly and characterization of nine tissues of Lonicera japonica to identify potential candidate genes involved in chlorogenic acid, luteolosides, and secoiridoid biosynthesis pathways
Article Snippet: .. RNA quality was assessed using Agilent Bioanalyzer 2100 (Agilent Technology, USA), and RNA samples with RNA integrity number (RIN) above 8 were used for cDNA library preparation. mRNA for each sample was isolated from the total RNA by using beads with oligo (dT), and were added with fragmentation buffer to shear mRNA into short fragments, which were then used as a template for the synthesis of first-strand cDNA using random hexamer primers. cDNA library for Illumina sequencing was prepared using SureSelect Strand specific RNA library kit (Agilent Technology, USA) according to the manufacturer’s instructions. .. Illumina sequencing and pre-processing of raw reads A cDNA library was sequenced using Illumina HiSeq™ 2000 sequencer (Illumina Inc., USA) to obtain paired-end reads with an average length of 101 bps. cDNA library preparation and sequencing were performed at Kazusa DNA Research Institute, Chiba, Japan.

Article Title: Combining Different mRNA Capture Methods to Analyze the Transcriptome: Analysis of the Xenopus laevis Transcriptome
Article Snippet: Selective capture or priming of reverse transcription using oligo-dT is the basis for the majority of cDNA library preparations and RNA-Seq library preparations. .. To compare the mRNAs sampled by oligo-dT and Cap-capture methods we prepared Illumina libraries from Xenopus laevis egg extracts arrested in metaphase of meiosis II (labeled Mitosis or M for the remainder of the paper) and extracts induced to enter interphase (IF) by the addition of calcium, which mimics fertilization induced calcium release[ ].

Article Title: Transcriptomic profiling of the salt-stress response in the wild recretohalophyte Reaumuria trigyna
Article Snippet: .. cDNA library construction and sequencing Beads with Oligo (dT) (Illumina) were used to isolate poly (A) + RNA from 20 μg of each RNA pool. .. Fragmentation buffer (Ambion, Austin, TX, USA) was added to break mRNA into short fragments.

Article Title: Functional characterization of three trehalase genes regulating the chitin metabolism pathway in rice brown planthopper using RNA interference
Article Snippet: Paragraph title: cDNA library construction and high-throughput sequencing ... The fragments apart from the 3′ cDNA fragments connected to Oligo (dT) were washed away, and the Illumina adaptor 1 was ligated to the 5′ cohesive ends.

Purification:

Article Title: An RNA Sequencing Transcriptome Analysis of Grasspea (Lathyrus sativus L.) and Development of SSR and KASP Markers
Article Snippet: Oligo-dT labeled magnetic beads (Illumina Inc., San Diego, USA) were used to combine the polyA of the mRNA for purifying the mRNA. .. Then, the short fragment RNAs were used to synthesize the first-strand cDNA with random primers, and this cDNA was transformed into double-strand cDNA using RnaseH and DNA polymerase I. Fragments of desirable lengths (200–300 bp) were purified by the QIAquick PCR Extraction Kit (Qiagen, Valencia, CA, USA).

Article Title: Functional characterization of three trehalase genes regulating the chitin metabolism pathway in rice brown planthopper using RNA interference
Article Snippet: The purified RNA samples were dissolved with Tris-base buffer, precipitated with ethanol, and resolubilised. .. The fragments apart from the 3′ cDNA fragments connected to Oligo (dT) were washed away, and the Illumina adaptor 1 was ligated to the 5′ cohesive ends.

Article Title: Genome-Wide Analysis of Polyadenylation Events in Schmidtea mediterranea
Article Snippet: The ligated RNA was then reverse transcribed using oligo dT (with an attached adapter sequence) and PCR amplified using primers from the TruSeq Illumina Small RNA library. .. The library was purified using Ampure XP beads and sequencing was performed on an Illumina HiSeq-1000.

Software:

Article Title: Novel chimeric transcript RRM2-c2orf48 promotes metastasis in nasopharyngeal carcinoma
Article Snippet: Beads bound to oligo(dT) were used to isolate poly(A) mRNA from total RNA. cDNA libraries were prepared and sequenced on the Illumina Hi-seq2000. .. The fusion gene detection analyzed using the SOAPfuse software.

Multiplex Assay:

Article Title: A Multiplex RNA-seq Strategy to Profile Poly(A+) RNA: Application to Analysis of Transcription Response and 3? End Formation
Article Snippet: Paragraph title: Experimental design for Multiplex Analysis of Poly(A)-linked Sequences (MAPS) ... Our strategy, depicted in , employs an oligo-dT linked to a specific sequencing primer (B) corresponding to the P7 sequence anchored on the surface of Illumina flowcells to prime cDNA synthesis.

Selection:

Article Title: Transcriptome sequencing in S?zary syndrome identifies S?zary cell and mycosis fungoides-associated lncRNAs and novel transcripts
Article Snippet: .. Briefly, oligo-dT–directed reverse transcription generated cDNAs corresponding to 3′ ends of polyadenylated transcripts; after linker ligation, size selection, and PCR amplification, the cDNAs were subjected to deep sequencing on the Illumina GAIIx platform with a raw read length of 36 bp. .. RNA-Seq reads were aligned to the human reference sequence National Center for Biotechnology Information (NCBI) build 36.1/hg18 with TopHat.

Article Title: A Multiplex RNA-seq Strategy to Profile Poly(A+) RNA: Application to Analysis of Transcription Response and 3? End Formation
Article Snippet: Our strategy, depicted in , employs an oligo-dT linked to a specific sequencing primer (B) corresponding to the P7 sequence anchored on the surface of Illumina flowcells to prime cDNA synthesis. .. The primer also carries a biotin moiety for solid phase selection.

Article Title: Combining Different mRNA Capture Methods to Analyze the Transcriptome: Analysis of the Xenopus laevis Transcriptome
Article Snippet: To determine if an alternative method of mRNA capture could be complementary to mRNA capture by oligo-dT selection we optimized a previously described method[ , ] to use recombinant human cap-binding protein eIF4E to capture mRNAs on the basis of the 5’ cap structure. .. To compare the mRNAs sampled by oligo-dT and Cap-capture methods we prepared Illumina libraries from Xenopus laevis egg extracts arrested in metaphase of meiosis II (labeled Mitosis or M for the remainder of the paper) and extracts induced to enter interphase (IF) by the addition of calcium, which mimics fertilization induced calcium release[ ].

Sample Prep:

Article Title: Transcriptome sequencing in S?zary syndrome identifies S?zary cell and mycosis fungoides-associated lncRNAs and novel transcripts
Article Snippet: RNA-Seq libraries were prepared with the mRNA Seq Sample Prep Kit (Illumina). mRNA was isolated by polyA selection from 1 to 2 μg of total RNA, fragmented, and randomly primed for reverse transcription, followed by second-strand cDNA synthesis. .. Briefly, oligo-dT–directed reverse transcription generated cDNAs corresponding to 3′ ends of polyadenylated transcripts; after linker ligation, size selection, and PCR amplification, the cDNAs were subjected to deep sequencing on the Illumina GAIIx platform with a raw read length of 36 bp.

Article Title: Integrating microRNA and mRNA expression profiling in Symbiodinium microadriaticum, a dinoflagellate symbiont of reef-building corals
Article Snippet: .. For mRNA sequencing, 2 × 100 bp paired-end reads for Illumina sequencing were generated from oligo(dT) selected total RNA using the Illumina TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA) according to manufacturer’s instructions. .. Sequence libraries for small RNAs were created with the Illumina TruSeq Small RNA Sample Prep Kit (Illumina, San Diego, CA) according to manufacturer’s instructions. mRNA sequencing libraries for the different conditions were multiplexed in equal quantities and ran on three lanes on the Illumina HiSeq 2000 platform producing a total of 302 million paired-end reads.

Article Title: Responses of the Housefly, Musca domestica, to the Hytrosavirus Replication: Impacts on Host's Vitellogenesis and Immunity
Article Snippet: .. Briefly, the mRNA was enriched from 1 μg of total RNA per sample using oligo-dT attached to magnetic beads and then subjected to thermal fragmentation using the elute, prime, and fragmentation mix from the Illumina TruSeq™ v2 RNA sample preparation kit. .. RNA fragments were then converted to double-stranded (ds)-cDNA using reverse transcriptase and random primers provided in the TruSeq RNA sample preparation kit.

Produced:

Article Title: Integrating microRNA and mRNA expression profiling in Symbiodinium microadriaticum, a dinoflagellate symbiont of reef-building corals
Article Snippet: For mRNA sequencing, 2 × 100 bp paired-end reads for Illumina sequencing were generated from oligo(dT) selected total RNA using the Illumina TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA) according to manufacturer’s instructions. .. Small RNA libraries were sequenced on 4 lanes on an Illumina Genome Analyzer IIx (GA2x) and produced a total of 137 million small RNA reads ≤ 32nt.

Activation Assay:

Article Title: RNA Sequencing of Laser-Capture Microdissected Compartments of the Maize Kernel Identifies Regulatory Modules Associated with Endosperm Cell Differentiation [OPEN]
Article Snippet: We selected this time point because it follows differentiation of the main cell types of the endosperm, which occurs at 4 to 6 , and precedes developmental programs associated with endosperm function, including the activation of storage product synthesis and deposition program, which initiates at ∼8 to 10 ( ; , ; ; ; ; ). .. Total RNA extracted from biological triplicates for the endosperm compartments and embryo, and single replicates of maternal compartments (22 samples) were reverse-transcribed to cDNA using oligo(dT) and random primers, amplified, and paired-end sequenced using an Illumina HiSequation 2000 platform.

High Throughput Screening Assay:

Article Title: Functional characterization of three trehalase genes regulating the chitin metabolism pathway in rice brown planthopper using RNA interference
Article Snippet: Paragraph title: cDNA library construction and high-throughput sequencing ... The fragments apart from the 3′ cDNA fragments connected to Oligo (dT) were washed away, and the Illumina adaptor 1 was ligated to the 5′ cohesive ends.

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    Illumina Inc oligo dt
    Comparison or mRNA-seq libraries from <t>oligo-dT</t> and Cap-captured mRNA. A-B. Reads from dT or Cap-capture prepared libraries were aligned to sequences consisting of the 5’ or 3’ 25% of Refseq <t>mRNAs.</t> Reads aligning to the 5’ and 3’ portions of the transcript are plotted. Blue line indicates a ratio of one. C. Reads from mRNA-seq libraries prepared using oligo-dT captured mRNAs from mitotic and interphase Xenopus egg extract were aligned to the Xenopus laevis Unigene database. Relative abundance of each mRNA in mitotic and interphase extracts is plotted. Red points highlight two-fold differences between mitotic and interphase samples. D. Same experiment as in panel C except that the mRNAs were purified using Cap-capture prior to library preparation. Red points highlight two-fold differences. E. Scatterplot of mRNA abundance in oligo-dT-captured and Cap-captured mRNA libraries. F. The ratio of reads per mRNA in mitotic and interphase extracts are plotted for oligo-dT captured mRNAs (X-axis, data from panel A) and cap-captured mRNAs (Y-axis data from panel B). The colored points correspond to mRNAs with known changes in poly-A tail length (cdk1, green; Eg2/aurora-a, orange; mos, red; Xlcl1, yellow). Quadrants highlight two-fold differences between samples.
    Oligo Dt, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 98/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Illumina Inc oligo dt purification
    Comparison of de novo transcriptome assemblies. A. Transcripts assembled by the Abyss assembler from <t>oligo-dT</t> and Cap-captured <t>mRNA</t> libraries were aligned to the X. tropicalis ENSEMBL annotated transcripts using BlastX. The number of ENSEMBL transcripts that were matched by each assembler:library pair are represented as a Venn Diagram. The number of transcripts present in each assembler:library pair are listed in Table 2. Because multiple sequences from each de novo assembly align to X. tropicalis genes these numbers are omitted from the figure for the sake of simplicity. B. Same comparison as in A, except that Velvet was used as the assembler instead of Abyss. C. Transcripts assembled by Abyss or Velvet from Cap-capture mRNA libraries were aligned to the X. tropicalis ENSEMBL transcripts using BLAT. Unique and common ENSEMBL transcripts are represented by a Venn Diagram D. Same comparison as in C, except that dT libraries are compared instead of Cap-captured libraries. E-H. Fraction of each ENSEMBL transcript covered by transcripts assembled using indicated assembler:library pair (from A-D) was calculated.
    Oligo Dt Purification, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/oligo dt purification/product/Illumina Inc
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    Comparison or mRNA-seq libraries from oligo-dT and Cap-captured mRNA. A-B. Reads from dT or Cap-capture prepared libraries were aligned to sequences consisting of the 5’ or 3’ 25% of Refseq mRNAs. Reads aligning to the 5’ and 3’ portions of the transcript are plotted. Blue line indicates a ratio of one. C. Reads from mRNA-seq libraries prepared using oligo-dT captured mRNAs from mitotic and interphase Xenopus egg extract were aligned to the Xenopus laevis Unigene database. Relative abundance of each mRNA in mitotic and interphase extracts is plotted. Red points highlight two-fold differences between mitotic and interphase samples. D. Same experiment as in panel C except that the mRNAs were purified using Cap-capture prior to library preparation. Red points highlight two-fold differences. E. Scatterplot of mRNA abundance in oligo-dT-captured and Cap-captured mRNA libraries. F. The ratio of reads per mRNA in mitotic and interphase extracts are plotted for oligo-dT captured mRNAs (X-axis, data from panel A) and cap-captured mRNAs (Y-axis data from panel B). The colored points correspond to mRNAs with known changes in poly-A tail length (cdk1, green; Eg2/aurora-a, orange; mos, red; Xlcl1, yellow). Quadrants highlight two-fold differences between samples.

    Journal: PLoS ONE

    Article Title: Combining Different mRNA Capture Methods to Analyze the Transcriptome: Analysis of the Xenopus laevis Transcriptome

    doi: 10.1371/journal.pone.0077700

    Figure Lengend Snippet: Comparison or mRNA-seq libraries from oligo-dT and Cap-captured mRNA. A-B. Reads from dT or Cap-capture prepared libraries were aligned to sequences consisting of the 5’ or 3’ 25% of Refseq mRNAs. Reads aligning to the 5’ and 3’ portions of the transcript are plotted. Blue line indicates a ratio of one. C. Reads from mRNA-seq libraries prepared using oligo-dT captured mRNAs from mitotic and interphase Xenopus egg extract were aligned to the Xenopus laevis Unigene database. Relative abundance of each mRNA in mitotic and interphase extracts is plotted. Red points highlight two-fold differences between mitotic and interphase samples. D. Same experiment as in panel C except that the mRNAs were purified using Cap-capture prior to library preparation. Red points highlight two-fold differences. E. Scatterplot of mRNA abundance in oligo-dT-captured and Cap-captured mRNA libraries. F. The ratio of reads per mRNA in mitotic and interphase extracts are plotted for oligo-dT captured mRNAs (X-axis, data from panel A) and cap-captured mRNAs (Y-axis data from panel B). The colored points correspond to mRNAs with known changes in poly-A tail length (cdk1, green; Eg2/aurora-a, orange; mos, red; Xlcl1, yellow). Quadrants highlight two-fold differences between samples.

    Article Snippet: To compare the mRNAs sampled by oligo-dT and Cap-capture methods we prepared Illumina libraries from Xenopus laevis egg extracts arrested in metaphase of meiosis II (labeled Mitosis or M for the remainder of the paper) and extracts induced to enter interphase (IF) by the addition of calcium, which mimics fertilization induced calcium release[ ].

    Techniques: Purification

    Poly-A tail analysis of selected mRNAs. mRNAs that exhibited changes in Mitosis:Interphase (M:IF) abundance ratios in oligo-dT-captured samples, but not in Cap-captured samples were analyzed for poly-A tail length using the ePAT assay and anchored TVN reverse transcription controls. A. Six mRNAs with high M:IF ratios (aurora-a, esco2, fbox5, stx11, march7, and hexim1) showed longer poly-A tails in mitotic extract. Two mRNA (setd8 and MGC83922) with a low M:IF ratio showed very modest changes in poly-A tail lengths between Mitosis and Interphase. Red asterix indicates the position of the prominent TVN PCR product that is quantified in B. B. The amount of PCR product contained in the TVN-RT PCR reactions (red asterix in A) were quantified. The ratio of the amount of PCR product in Mitotic to Interphase extracts is presented in the first line. The ratio of each mRNA in Mitotic and Interphase extracts as determined by RNA-seq is presented below the PCR derived ratios for comparison. In addition five mRNAs with high M:IF ratios (aurora-1, fbox5, stx11, march7, and hexim1) had increased amounts of minimal poly-A tail PCR products in mitosis compared to interphase in TVN controls (quantified below gel) while both setd8 and MGC88922 had higher levels of TVN PCR products in interphase compared to mitotic extracts TVN PCR products indicate mRNAs with poly-A tails of 18 As. Line traces of ePAT PCR reactions presented in panel A. Black lines indicate traces from mitotic extract and red lines indicate traces from interaphse extract. D. Semi-quantitative PCR for each of the mRNAs tested in A was performed on RNA from mitotic and interphase extracts. Random hexamers were used to prime reverse transcription for these reactions. A second experiment showing very similar results is present in Figure S2.

    Journal: PLoS ONE

    Article Title: Combining Different mRNA Capture Methods to Analyze the Transcriptome: Analysis of the Xenopus laevis Transcriptome

    doi: 10.1371/journal.pone.0077700

    Figure Lengend Snippet: Poly-A tail analysis of selected mRNAs. mRNAs that exhibited changes in Mitosis:Interphase (M:IF) abundance ratios in oligo-dT-captured samples, but not in Cap-captured samples were analyzed for poly-A tail length using the ePAT assay and anchored TVN reverse transcription controls. A. Six mRNAs with high M:IF ratios (aurora-a, esco2, fbox5, stx11, march7, and hexim1) showed longer poly-A tails in mitotic extract. Two mRNA (setd8 and MGC83922) with a low M:IF ratio showed very modest changes in poly-A tail lengths between Mitosis and Interphase. Red asterix indicates the position of the prominent TVN PCR product that is quantified in B. B. The amount of PCR product contained in the TVN-RT PCR reactions (red asterix in A) were quantified. The ratio of the amount of PCR product in Mitotic to Interphase extracts is presented in the first line. The ratio of each mRNA in Mitotic and Interphase extracts as determined by RNA-seq is presented below the PCR derived ratios for comparison. In addition five mRNAs with high M:IF ratios (aurora-1, fbox5, stx11, march7, and hexim1) had increased amounts of minimal poly-A tail PCR products in mitosis compared to interphase in TVN controls (quantified below gel) while both setd8 and MGC88922 had higher levels of TVN PCR products in interphase compared to mitotic extracts TVN PCR products indicate mRNAs with poly-A tails of 18 As. Line traces of ePAT PCR reactions presented in panel A. Black lines indicate traces from mitotic extract and red lines indicate traces from interaphse extract. D. Semi-quantitative PCR for each of the mRNAs tested in A was performed on RNA from mitotic and interphase extracts. Random hexamers were used to prime reverse transcription for these reactions. A second experiment showing very similar results is present in Figure S2.

    Article Snippet: To compare the mRNAs sampled by oligo-dT and Cap-capture methods we prepared Illumina libraries from Xenopus laevis egg extracts arrested in metaphase of meiosis II (labeled Mitosis or M for the remainder of the paper) and extracts induced to enter interphase (IF) by the addition of calcium, which mimics fertilization induced calcium release[ ].

    Techniques: Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, RNA Sequencing Assay, Derivative Assay, Real-time Polymerase Chain Reaction

    Analysis of RNA samples taken during the granule enrichment. ( A ) Northern blots. Northern gels loaded with total RNA samples were probed for total mRNAs and for 18S rRNA. SL = spliced leader RNA. ( B ) The percentage of mRNA in the ‘granule’ fraction of non-starved and starved cells was quantified from northern blots of three independent experiments. Note that this percentage is calculated based on total mRNA being the sum of SN1, SN2, SN3, SN4 and P4; ‘T’ is mRNA prepared in parallel as a control for mRNA quality and is not equivalent to the other samples. ( C and D ) Starved trypanosomes were stained for total mRNA with a fluorescent oligo antisense to the mini-exon sequence (RNA FISH): one representative cell is shown in C. The fraction of fluorescence in granules in comparison to total fluorescence was quantified from Z-stack projections of 49 cells (all were in a early cell cycle stage prior to the division of their kinetoplast and nucleus). Non-starved trypanosomes had no visible granules (see Figures 8 ) and hybridization with an oligo sense to the spliced leader sequence (negative control) gave a 14-fold weaker total signal (data not shown). This experiment is one representative of several.

    Journal: Nucleic Acids Research

    Article Title: Novel insights into RNP granules by employing the trypanosome's microtubule skeleton as a molecular sieve

    doi: 10.1093/nar/gkv731

    Figure Lengend Snippet: Analysis of RNA samples taken during the granule enrichment. ( A ) Northern blots. Northern gels loaded with total RNA samples were probed for total mRNAs and for 18S rRNA. SL = spliced leader RNA. ( B ) The percentage of mRNA in the ‘granule’ fraction of non-starved and starved cells was quantified from northern blots of three independent experiments. Note that this percentage is calculated based on total mRNA being the sum of SN1, SN2, SN3, SN4 and P4; ‘T’ is mRNA prepared in parallel as a control for mRNA quality and is not equivalent to the other samples. ( C and D ) Starved trypanosomes were stained for total mRNA with a fluorescent oligo antisense to the mini-exon sequence (RNA FISH): one representative cell is shown in C. The fraction of fluorescence in granules in comparison to total fluorescence was quantified from Z-stack projections of 49 cells (all were in a early cell cycle stage prior to the division of their kinetoplast and nucleus). Non-starved trypanosomes had no visible granules (see Figures 8 ) and hybridization with an oligo sense to the spliced leader sequence (negative control) gave a 14-fold weaker total signal (data not shown). This experiment is one representative of several.

    Article Snippet: To analyse mRNAs of starved cells, total RNA and RNA from fraction P4 of two independent experiments were enriched for mRNAs by oligo(dT) affinity and analysed by Illumina RNA sequencing.

    Techniques: Northern Blot, Staining, Sequencing, Fluorescence In Situ Hybridization, Fluorescence, Hybridization, Negative Control

    Comparison of de novo transcriptome assemblies. A. Transcripts assembled by the Abyss assembler from oligo-dT and Cap-captured mRNA libraries were aligned to the X. tropicalis ENSEMBL annotated transcripts using BlastX. The number of ENSEMBL transcripts that were matched by each assembler:library pair are represented as a Venn Diagram. The number of transcripts present in each assembler:library pair are listed in Table 2. Because multiple sequences from each de novo assembly align to X. tropicalis genes these numbers are omitted from the figure for the sake of simplicity. B. Same comparison as in A, except that Velvet was used as the assembler instead of Abyss. C. Transcripts assembled by Abyss or Velvet from Cap-capture mRNA libraries were aligned to the X. tropicalis ENSEMBL transcripts using BLAT. Unique and common ENSEMBL transcripts are represented by a Venn Diagram D. Same comparison as in C, except that dT libraries are compared instead of Cap-captured libraries. E-H. Fraction of each ENSEMBL transcript covered by transcripts assembled using indicated assembler:library pair (from A-D) was calculated.

    Journal: PLoS ONE

    Article Title: Combining Different mRNA Capture Methods to Analyze the Transcriptome: Analysis of the Xenopus laevis Transcriptome

    doi: 10.1371/journal.pone.0077700

    Figure Lengend Snippet: Comparison of de novo transcriptome assemblies. A. Transcripts assembled by the Abyss assembler from oligo-dT and Cap-captured mRNA libraries were aligned to the X. tropicalis ENSEMBL annotated transcripts using BlastX. The number of ENSEMBL transcripts that were matched by each assembler:library pair are represented as a Venn Diagram. The number of transcripts present in each assembler:library pair are listed in Table 2. Because multiple sequences from each de novo assembly align to X. tropicalis genes these numbers are omitted from the figure for the sake of simplicity. B. Same comparison as in A, except that Velvet was used as the assembler instead of Abyss. C. Transcripts assembled by Abyss or Velvet from Cap-capture mRNA libraries were aligned to the X. tropicalis ENSEMBL transcripts using BLAT. Unique and common ENSEMBL transcripts are represented by a Venn Diagram D. Same comparison as in C, except that dT libraries are compared instead of Cap-captured libraries. E-H. Fraction of each ENSEMBL transcript covered by transcripts assembled using indicated assembler:library pair (from A-D) was calculated.

    Article Snippet: Supporting Information Assessment of mRNA enrichment by oligo-dT and cap-capture. mRNA was captured from total extract using either oligo-dT purification or cap-capture purification.

    Techniques:

    Comparison or mRNA-seq libraries from oligo-dT and Cap-captured mRNA. A-B. Reads from dT or Cap-capture prepared libraries were aligned to sequences consisting of the 5’ or 3’ 25% of Refseq mRNAs. Reads aligning to the 5’ and 3’ portions of the transcript are plotted. Blue line indicates a ratio of one. C. Reads from mRNA-seq libraries prepared using oligo-dT captured mRNAs from mitotic and interphase Xenopus egg extract were aligned to the Xenopus laevis Unigene database. Relative abundance of each mRNA in mitotic and interphase extracts is plotted. Red points highlight two-fold differences between mitotic and interphase samples. D. Same experiment as in panel C except that the mRNAs were purified using Cap-capture prior to library preparation. Red points highlight two-fold differences. E. Scatterplot of mRNA abundance in oligo-dT-captured and Cap-captured mRNA libraries. F. The ratio of reads per mRNA in mitotic and interphase extracts are plotted for oligo-dT captured mRNAs (X-axis, data from panel A) and cap-captured mRNAs (Y-axis data from panel B). The colored points correspond to mRNAs with known changes in poly-A tail length (cdk1, green; Eg2/aurora-a, orange; mos, red; Xlcl1, yellow). Quadrants highlight two-fold differences between samples.

    Journal: PLoS ONE

    Article Title: Combining Different mRNA Capture Methods to Analyze the Transcriptome: Analysis of the Xenopus laevis Transcriptome

    doi: 10.1371/journal.pone.0077700

    Figure Lengend Snippet: Comparison or mRNA-seq libraries from oligo-dT and Cap-captured mRNA. A-B. Reads from dT or Cap-capture prepared libraries were aligned to sequences consisting of the 5’ or 3’ 25% of Refseq mRNAs. Reads aligning to the 5’ and 3’ portions of the transcript are plotted. Blue line indicates a ratio of one. C. Reads from mRNA-seq libraries prepared using oligo-dT captured mRNAs from mitotic and interphase Xenopus egg extract were aligned to the Xenopus laevis Unigene database. Relative abundance of each mRNA in mitotic and interphase extracts is plotted. Red points highlight two-fold differences between mitotic and interphase samples. D. Same experiment as in panel C except that the mRNAs were purified using Cap-capture prior to library preparation. Red points highlight two-fold differences. E. Scatterplot of mRNA abundance in oligo-dT-captured and Cap-captured mRNA libraries. F. The ratio of reads per mRNA in mitotic and interphase extracts are plotted for oligo-dT captured mRNAs (X-axis, data from panel A) and cap-captured mRNAs (Y-axis data from panel B). The colored points correspond to mRNAs with known changes in poly-A tail length (cdk1, green; Eg2/aurora-a, orange; mos, red; Xlcl1, yellow). Quadrants highlight two-fold differences between samples.

    Article Snippet: Supporting Information Assessment of mRNA enrichment by oligo-dT and cap-capture. mRNA was captured from total extract using either oligo-dT purification or cap-capture purification.

    Techniques: Purification

    Poly-A tail analysis of selected mRNAs. mRNAs that exhibited changes in Mitosis:Interphase (M:IF) abundance ratios in oligo-dT-captured samples, but not in Cap-captured samples were analyzed for poly-A tail length using the ePAT assay and anchored TVN reverse transcription controls. A. Six mRNAs with high M:IF ratios (aurora-a, esco2, fbox5, stx11, march7, and hexim1) showed longer poly-A tails in mitotic extract. Two mRNA (setd8 and MGC83922) with a low M:IF ratio showed very modest changes in poly-A tail lengths between Mitosis and Interphase. Red asterix indicates the position of the prominent TVN PCR product that is quantified in B. B. The amount of PCR product contained in the TVN-RT PCR reactions (red asterix in A) were quantified. The ratio of the amount of PCR product in Mitotic to Interphase extracts is presented in the first line. The ratio of each mRNA in Mitotic and Interphase extracts as determined by RNA-seq is presented below the PCR derived ratios for comparison. In addition five mRNAs with high M:IF ratios (aurora-1, fbox5, stx11, march7, and hexim1) had increased amounts of minimal poly-A tail PCR products in mitosis compared to interphase in TVN controls (quantified below gel) while both setd8 and MGC88922 had higher levels of TVN PCR products in interphase compared to mitotic extracts TVN PCR products indicate mRNAs with poly-A tails of 18 As. Line traces of ePAT PCR reactions presented in panel A. Black lines indicate traces from mitotic extract and red lines indicate traces from interaphse extract. D. Semi-quantitative PCR for each of the mRNAs tested in A was performed on RNA from mitotic and interphase extracts. Random hexamers were used to prime reverse transcription for these reactions. A second experiment showing very similar results is present in Figure S2.

    Journal: PLoS ONE

    Article Title: Combining Different mRNA Capture Methods to Analyze the Transcriptome: Analysis of the Xenopus laevis Transcriptome

    doi: 10.1371/journal.pone.0077700

    Figure Lengend Snippet: Poly-A tail analysis of selected mRNAs. mRNAs that exhibited changes in Mitosis:Interphase (M:IF) abundance ratios in oligo-dT-captured samples, but not in Cap-captured samples were analyzed for poly-A tail length using the ePAT assay and anchored TVN reverse transcription controls. A. Six mRNAs with high M:IF ratios (aurora-a, esco2, fbox5, stx11, march7, and hexim1) showed longer poly-A tails in mitotic extract. Two mRNA (setd8 and MGC83922) with a low M:IF ratio showed very modest changes in poly-A tail lengths between Mitosis and Interphase. Red asterix indicates the position of the prominent TVN PCR product that is quantified in B. B. The amount of PCR product contained in the TVN-RT PCR reactions (red asterix in A) were quantified. The ratio of the amount of PCR product in Mitotic to Interphase extracts is presented in the first line. The ratio of each mRNA in Mitotic and Interphase extracts as determined by RNA-seq is presented below the PCR derived ratios for comparison. In addition five mRNAs with high M:IF ratios (aurora-1, fbox5, stx11, march7, and hexim1) had increased amounts of minimal poly-A tail PCR products in mitosis compared to interphase in TVN controls (quantified below gel) while both setd8 and MGC88922 had higher levels of TVN PCR products in interphase compared to mitotic extracts TVN PCR products indicate mRNAs with poly-A tails of 18 As. Line traces of ePAT PCR reactions presented in panel A. Black lines indicate traces from mitotic extract and red lines indicate traces from interaphse extract. D. Semi-quantitative PCR for each of the mRNAs tested in A was performed on RNA from mitotic and interphase extracts. Random hexamers were used to prime reverse transcription for these reactions. A second experiment showing very similar results is present in Figure S2.

    Article Snippet: Supporting Information Assessment of mRNA enrichment by oligo-dT and cap-capture. mRNA was captured from total extract using either oligo-dT purification or cap-capture purification.

    Techniques: Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, RNA Sequencing Assay, Derivative Assay, Real-time Polymerase Chain Reaction