Structured Review

Illumina Inc complementary dna cdna
HLA-DQB2 expression is identified on blood BRAF-V600E + CD1c + mDCs in patients with high-risk (HR) LCH. (A) Genomic <t>DNA</t> was isolated from peripheral blood specimens from LCH patients (N = 11; n = 3 for high-risk multisystem, n = 3 for low-risk [LR] multisystem, n = 5 for low-risk single lesion) with BRAF V600E + lesions and healthy donors (N = 11). The percentage of circulating cells with BRAF V600 E As expected from previous studies, BRAF V600E expression was detected at low levels in PBMCs from patients with BRAF V600E + Technical duplicates were used in this experiment. LCH patients’ PBMC samples used in the study are listed in supplemental Table 1b. Blue dot, Patient LCH 0019; red dot, patient LCH 0020; green dot, patient LCH 0021. (B) RNA from peripheral blood specimens from the same set of LCH patients as in panel A was extracted and <t>cDNA</t> was amplified, and then the HLA-DQB2 expression was determined by qPCR (normalized to GAPDH mRNA expression). HLA-DQB2 expression was specifically detected in PBMCs from the same patients with detectable BRAF V600E + PBMCs. Technical duplicates were used in this experiment. Blue dot, Patient LCH 0019; red dot, patient LCH 0020; green dot, patient LCH 0021). (C) Representative dot plots showing identification of CD1c + mDCs (green gate) within HLA-DR + cells from PBMCs of an HR LCH patient. Overlay histograms show HLA-DQB2 expression in LCH lesion CD1c + mDCs (green) compared with control (gray). HLA-DQB2 expression was detectable on some CD1c + mDCs. Representative dot plots showing identification of CD1c + mDCs (green gate) from PBMCs of a healthy donor were illustrated in supplemental Figure 8. No HLA-DQB2 expression was detectable on CD1c + mDCs from PBMCs of a healthy donor. (D) Genomic DNA from unsorted and sorted cells from PBMCs of high-risk LCH patients (N = 3) with BRAF V600E + lesions was isolated and amplified, and the percentage of cells with BRAF V600E allele was determined by qPCR. As demonstrated in previous studies, many lineages have the potential to carry the BRAF Technical duplicates were used in this experiment. Blue dot, Patient LCH 0019; red dot, patient LCH 0020; green dot, patient LCH 0021. (E) Genomic DNA from CD1c + mDCs (HLA-DQB2 - and HLA-DQB2 + ) from PBMCs of high-risk LCH patients (N = 3) was isolated and amplified, and the percentage of cells with BRAF V600E allele was determined by qPCR. BRAF V600E was highly enriched in the HLA-DQB2 + CD1c + mDC population. Technical duplicates were used in this experiment. Blue dot, Patient LCH 0019; red dot, patient LCH 0020; green dot, patient LCH 0021.
Complementary Dna Cdna, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Circulating CD1c+ myeloid dendritic cells are potential precursors to LCH lesion CD1a+CD207+ cells"

Article Title: Circulating CD1c+ myeloid dendritic cells are potential precursors to LCH lesion CD1a+CD207+ cells

Journal: Blood Advances

doi: 10.1182/bloodadvances.2019000488

HLA-DQB2 expression is identified on blood BRAF-V600E + CD1c + mDCs in patients with high-risk (HR) LCH. (A) Genomic DNA was isolated from peripheral blood specimens from LCH patients (N = 11; n = 3 for high-risk multisystem, n = 3 for low-risk [LR] multisystem, n = 5 for low-risk single lesion) with BRAF V600E + lesions and healthy donors (N = 11). The percentage of circulating cells with BRAF V600 E As expected from previous studies, BRAF V600E expression was detected at low levels in PBMCs from patients with BRAF V600E + Technical duplicates were used in this experiment. LCH patients’ PBMC samples used in the study are listed in supplemental Table 1b. Blue dot, Patient LCH 0019; red dot, patient LCH 0020; green dot, patient LCH 0021. (B) RNA from peripheral blood specimens from the same set of LCH patients as in panel A was extracted and cDNA was amplified, and then the HLA-DQB2 expression was determined by qPCR (normalized to GAPDH mRNA expression). HLA-DQB2 expression was specifically detected in PBMCs from the same patients with detectable BRAF V600E + PBMCs. Technical duplicates were used in this experiment. Blue dot, Patient LCH 0019; red dot, patient LCH 0020; green dot, patient LCH 0021). (C) Representative dot plots showing identification of CD1c + mDCs (green gate) within HLA-DR + cells from PBMCs of an HR LCH patient. Overlay histograms show HLA-DQB2 expression in LCH lesion CD1c + mDCs (green) compared with control (gray). HLA-DQB2 expression was detectable on some CD1c + mDCs. Representative dot plots showing identification of CD1c + mDCs (green gate) from PBMCs of a healthy donor were illustrated in supplemental Figure 8. No HLA-DQB2 expression was detectable on CD1c + mDCs from PBMCs of a healthy donor. (D) Genomic DNA from unsorted and sorted cells from PBMCs of high-risk LCH patients (N = 3) with BRAF V600E + lesions was isolated and amplified, and the percentage of cells with BRAF V600E allele was determined by qPCR. As demonstrated in previous studies, many lineages have the potential to carry the BRAF Technical duplicates were used in this experiment. Blue dot, Patient LCH 0019; red dot, patient LCH 0020; green dot, patient LCH 0021. (E) Genomic DNA from CD1c + mDCs (HLA-DQB2 - and HLA-DQB2 + ) from PBMCs of high-risk LCH patients (N = 3) was isolated and amplified, and the percentage of cells with BRAF V600E allele was determined by qPCR. BRAF V600E was highly enriched in the HLA-DQB2 + CD1c + mDC population. Technical duplicates were used in this experiment. Blue dot, Patient LCH 0019; red dot, patient LCH 0020; green dot, patient LCH 0021.
Figure Legend Snippet: HLA-DQB2 expression is identified on blood BRAF-V600E + CD1c + mDCs in patients with high-risk (HR) LCH. (A) Genomic DNA was isolated from peripheral blood specimens from LCH patients (N = 11; n = 3 for high-risk multisystem, n = 3 for low-risk [LR] multisystem, n = 5 for low-risk single lesion) with BRAF V600E + lesions and healthy donors (N = 11). The percentage of circulating cells with BRAF V600 E As expected from previous studies, BRAF V600E expression was detected at low levels in PBMCs from patients with BRAF V600E + Technical duplicates were used in this experiment. LCH patients’ PBMC samples used in the study are listed in supplemental Table 1b. Blue dot, Patient LCH 0019; red dot, patient LCH 0020; green dot, patient LCH 0021. (B) RNA from peripheral blood specimens from the same set of LCH patients as in panel A was extracted and cDNA was amplified, and then the HLA-DQB2 expression was determined by qPCR (normalized to GAPDH mRNA expression). HLA-DQB2 expression was specifically detected in PBMCs from the same patients with detectable BRAF V600E + PBMCs. Technical duplicates were used in this experiment. Blue dot, Patient LCH 0019; red dot, patient LCH 0020; green dot, patient LCH 0021). (C) Representative dot plots showing identification of CD1c + mDCs (green gate) within HLA-DR + cells from PBMCs of an HR LCH patient. Overlay histograms show HLA-DQB2 expression in LCH lesion CD1c + mDCs (green) compared with control (gray). HLA-DQB2 expression was detectable on some CD1c + mDCs. Representative dot plots showing identification of CD1c + mDCs (green gate) from PBMCs of a healthy donor were illustrated in supplemental Figure 8. No HLA-DQB2 expression was detectable on CD1c + mDCs from PBMCs of a healthy donor. (D) Genomic DNA from unsorted and sorted cells from PBMCs of high-risk LCH patients (N = 3) with BRAF V600E + lesions was isolated and amplified, and the percentage of cells with BRAF V600E allele was determined by qPCR. As demonstrated in previous studies, many lineages have the potential to carry the BRAF Technical duplicates were used in this experiment. Blue dot, Patient LCH 0019; red dot, patient LCH 0020; green dot, patient LCH 0021. (E) Genomic DNA from CD1c + mDCs (HLA-DQB2 - and HLA-DQB2 + ) from PBMCs of high-risk LCH patients (N = 3) was isolated and amplified, and the percentage of cells with BRAF V600E allele was determined by qPCR. BRAF V600E was highly enriched in the HLA-DQB2 + CD1c + mDC population. Technical duplicates were used in this experiment. Blue dot, Patient LCH 0019; red dot, patient LCH 0020; green dot, patient LCH 0021.

Techniques Used: Expressing, Isolation, Amplification, Real-time Polymerase Chain Reaction

2) Product Images from "scDual-Seq: mapping the gene regulatory program of Salmonella infection by host and pathogen single-cell RNA-sequencing"

Article Title: scDual-Seq: mapping the gene regulatory program of Salmonella infection by host and pathogen single-cell RNA-sequencing

Journal: Genome Biology

doi: 10.1186/s13059-017-1340-x

A single-cell RNA-sequencing approach to studying host–pathogen interaction. a Heterogeneity of outcomes of intracellular infection is due to both Salmonella and macrophage states. scDual-Seq simultaneously produces the transcriptome of both the host and the pathogen and allows the identification of cellular subpopulations during infection. b Schematic of the scDual-Seq method. Reverse transcription is primed using random hexamers, followed by RNase treatment and 3’ polyA tailing. The second strand is synthesized using the CEL-Seq2 barcoded primers (see “ Methods ”). The samples are pooled together before the complementary DNA (cDNA) undergoes linear amplification by in vitro transcription. The amplified RNA is then reverse transcribed using a random primer with an overhang of the sequence complementary to the Illumina 3’ adaptor. cDNA with both Illumina adaptors are selected by polymerase chain reaction and the DNA library is sequenced using paired-end Illumina sequencing. c Mean number of unique transcripts identified across five technical replicates, for mouse ( black ) and Salmonella ( red ). Circles and error bars represent the mean and standard deviation. d Plot between the expression of the two technical replicates of 10 pg mouse RNA and 10 pg Salmonella RNA. e Boxplots indicating the correlation coefficients across replicates with the sum expression of all 20 samples for mouse and for five replicates in each dilution for Salmonella . Mouse indicated in black , Salmonella dilutions indicated in red
Figure Legend Snippet: A single-cell RNA-sequencing approach to studying host–pathogen interaction. a Heterogeneity of outcomes of intracellular infection is due to both Salmonella and macrophage states. scDual-Seq simultaneously produces the transcriptome of both the host and the pathogen and allows the identification of cellular subpopulations during infection. b Schematic of the scDual-Seq method. Reverse transcription is primed using random hexamers, followed by RNase treatment and 3’ polyA tailing. The second strand is synthesized using the CEL-Seq2 barcoded primers (see “ Methods ”). The samples are pooled together before the complementary DNA (cDNA) undergoes linear amplification by in vitro transcription. The amplified RNA is then reverse transcribed using a random primer with an overhang of the sequence complementary to the Illumina 3’ adaptor. cDNA with both Illumina adaptors are selected by polymerase chain reaction and the DNA library is sequenced using paired-end Illumina sequencing. c Mean number of unique transcripts identified across five technical replicates, for mouse ( black ) and Salmonella ( red ). Circles and error bars represent the mean and standard deviation. d Plot between the expression of the two technical replicates of 10 pg mouse RNA and 10 pg Salmonella RNA. e Boxplots indicating the correlation coefficients across replicates with the sum expression of all 20 samples for mouse and for five replicates in each dilution for Salmonella . Mouse indicated in black , Salmonella dilutions indicated in red

Techniques Used: RNA Sequencing Assay, Infection, Synthesized, Amplification, In Vitro, Sequencing, Polymerase Chain Reaction, Standard Deviation, Expressing

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Article Snippet: .. cDNA preparation and transcriptome sequencing Mental glands were collected from male P. shermani at six time points approximately every 3 weeks during 2010 (5/29, 6/19, 7/10, 8/1, 8/21, and 9/11). ..

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    Illumina Inc cdna quantification
    Effect of the GC content and ERCC length on the estimation of the ERCC <t>cDNA</t> abundance with the <t>ONT</t> MinION platform. The figures present deviations of the ERCC expression level estimates with the ONT MinION platform from the Ambion RNA molecular counts ( A,C ) or from the Illumina HiSeq 2500/MiSeq estimated cDNA abundance ( B,D ) as a function of the GC content ( A,B ) and the ERCC length ( C,D ). We plot the log2 ratio of observed (ONT MinION) to expected (Ambion, Illumina) read counts for the ERCC spike-ins (y-axis, log) for each of the samples relative to their length or GC content (x-axis). Due to the variable sequencing depth from each ERCC MinION experiment, each point is the average value from different MinION flow cell runs if at least 5 reads have been detected for this point in the corresponding MinION runs. The points are colored differently based on the number of flow cell runs in which they were detected (red, green, cyan, purple correspond to values derived from one, two, three or four flow cell runs respectively). The standard deviation is also presented for points with values from two or more MinION runs.
    Cdna Quantification, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc ercc cdna molecules
    Effect of the GC content and <t>ERCC</t> length on the estimation of the ERCC <t>cDNA</t> abundance with the ONT MinION platform. The figures present deviations of the ERCC expression level estimates with the ONT MinION platform from the Ambion RNA molecular counts ( A,C ) or from the Illumina HiSeq 2500/MiSeq estimated cDNA abundance ( B,D ) as a function of the GC content ( A,B ) and the ERCC length ( C,D ). We plot the log2 ratio of observed (ONT MinION) to expected (Ambion, Illumina) read counts for the ERCC spike-ins (y-axis, log) for each of the samples relative to their length or GC content (x-axis). Due to the variable sequencing depth from each ERCC MinION experiment, each point is the average value from different MinION flow cell runs if at least 5 reads have been detected for this point in the corresponding MinION runs. The points are colored differently based on the number of flow cell runs in which they were detected (red, green, cyan, purple correspond to values derived from one, two, three or four flow cell runs respectively). The standard deviation is also presented for points with values from two or more MinION runs.
    Ercc Cdna Molecules, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc solexa illumina cdna sequencing
    Relative proportion of sRNA candidates in different classes . (a) 454 sequencing: distribution of reads mapped to the S. meliloti 1021 genome and distribution of the analyzed contigs according to the general classification (Figure 3). Left circle diagram: light colored (I) and colored (II), number of reads derived from sample 1 and 2. Reads in sample 1 and 2: non-mapped, 48,159 and 57,964; rRNA genes, 67,891 and 176,848; tRNA genes, 188,121 and 79,789; repeats, 3,029 and 6,206; IGRs or ORFs, 77,326 and 140,702. Right circle diagram: light colored (I), colored (II) and dark colored (I+II) represent the number of RNA candidates derived from sample 1, sample 2, and both samples, respectively: trans-encoded sRNAs, 28, 38, 85; cis-encoded antisense sRNAs, 9, 52, 35; mRNA leader transcripts, 46, 151, 181; sense sRNAs 28, 363, 56; ORFs 0, 4, 4. (b) <t>Illumina/Solexa</t> sequencing: Distribution of reads mapped to the S. meliloti 1021 genome. Reads: non-mapped, 1,179,722; rRNA genes, 3,405,289; tRNA genes, 1,058,534; repeats, 111,355; IGR and ORFs, 711,851. Dark green segment: contigs for 44 putative trans-encoded sRNAs. (c) Microarray-based analysis and (d) Affymetrix Symbiosis Chip-based analysis: distribution of sRNA candidates. Segment numbers represent subtypes. Microarray data: type 1 and 2 trans-encoded sRNAs, 264 and 721 candidates; type 1, 2 and 3 cis-encoded antisense sRNAs, 25, 587 and 59; mRNA leader transcripts, 250. Affymetrix Symbiosis Chip data: type 1 and 2 trans-encoded sRNAs, 60 and 174; type 1, 2 and 3 cis-encoded antisense sRNAs, 3, 4 and 27; mRNA leader, 112.
    Solexa Illumina Cdna Sequencing, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc cyclic phosphate cdna synthesis
    Viral RNA fragments produced by RNase L and RNase A. HCV and PV RNAs were incubated with RNase L and RNase A to produce RNA fragments for 2′, <t>3′-cyclic</t> phosphate <t>cDNA</t> synthesis and sequencing. Agarose gel electrophoresis and ethidium bromide staining revealed the size of viral RNA fragments. ( A ) Diagram of HCV and PV RNAs. HCV RNA is 9648 bases long. PV RNA is 7500 bases long. ( B ) Viral RNAs incubated with RNase L. HCV and PV RNAs were incubated with RNase L for 20 min in the absence of 2-5A (no 2-5A), or with RNase L and 2-5A for 0, 2.5, 5, 10 and 20 min. ( C ) Viral RNAs incubated with RNase A. HCV and PV RNAs were incubated for 20 min in the absence of RNase A (−), and the presence of RNase A for 0, 2.5, 5, 10 and 20 min.
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    Effect of the GC content and ERCC length on the estimation of the ERCC cDNA abundance with the ONT MinION platform. The figures present deviations of the ERCC expression level estimates with the ONT MinION platform from the Ambion RNA molecular counts ( A,C ) or from the Illumina HiSeq 2500/MiSeq estimated cDNA abundance ( B,D ) as a function of the GC content ( A,B ) and the ERCC length ( C,D ). We plot the log2 ratio of observed (ONT MinION) to expected (Ambion, Illumina) read counts for the ERCC spike-ins (y-axis, log) for each of the samples relative to their length or GC content (x-axis). Due to the variable sequencing depth from each ERCC MinION experiment, each point is the average value from different MinION flow cell runs if at least 5 reads have been detected for this point in the corresponding MinION runs. The points are colored differently based on the number of flow cell runs in which they were detected (red, green, cyan, purple correspond to values derived from one, two, three or four flow cell runs respectively). The standard deviation is also presented for points with values from two or more MinION runs.

    Journal: Scientific Reports

    Article Title: Benchmarking of the Oxford Nanopore MinION sequencing for quantitative and qualitative assessment of cDNA populations

    doi: 10.1038/srep31602

    Figure Lengend Snippet: Effect of the GC content and ERCC length on the estimation of the ERCC cDNA abundance with the ONT MinION platform. The figures present deviations of the ERCC expression level estimates with the ONT MinION platform from the Ambion RNA molecular counts ( A,C ) or from the Illumina HiSeq 2500/MiSeq estimated cDNA abundance ( B,D ) as a function of the GC content ( A,B ) and the ERCC length ( C,D ). We plot the log2 ratio of observed (ONT MinION) to expected (Ambion, Illumina) read counts for the ERCC spike-ins (y-axis, log) for each of the samples relative to their length or GC content (x-axis). Due to the variable sequencing depth from each ERCC MinION experiment, each point is the average value from different MinION flow cell runs if at least 5 reads have been detected for this point in the corresponding MinION runs. The points are colored differently based on the number of flow cell runs in which they were detected (red, green, cyan, purple correspond to values derived from one, two, three or four flow cell runs respectively). The standard deviation is also presented for points with values from two or more MinION runs.

    Article Snippet: For cDNA quantification, the ONT MinION platform can be used instead of the Illumina platform as it provides comparable quantitative data.

    Techniques: Expressing, Sequencing, Flow Cytometry, Derivative Assay, Standard Deviation

    Estimation of the ERCC cDNA abundance with the ONT MinION platform. We compared the ERCC cDNA abundance estimated from the ONT MinION ( A ) and the Illumina HiSeq 2500 or MiSeq platforms ( B ) against the expected number of RNA molecules as provided from the manufacturer (Ambion). The template reads from the ERCC MinION experiments number 1, 2, 3, 4 were pooled together and used for ( A ). Similarly, the corresponding Illumina data were pooled together for ( B ). The Illumina molecular counts data were derived using 5′ molecular tags at the RT step as described in material and methods. The total number of molecules presented on the x-axis corresponds to 3.5 pgs of ERCC RNA. In both axes the log10 transformation of the original count number is used.

    Journal: Scientific Reports

    Article Title: Benchmarking of the Oxford Nanopore MinION sequencing for quantitative and qualitative assessment of cDNA populations

    doi: 10.1038/srep31602

    Figure Lengend Snippet: Estimation of the ERCC cDNA abundance with the ONT MinION platform. We compared the ERCC cDNA abundance estimated from the ONT MinION ( A ) and the Illumina HiSeq 2500 or MiSeq platforms ( B ) against the expected number of RNA molecules as provided from the manufacturer (Ambion). The template reads from the ERCC MinION experiments number 1, 2, 3, 4 were pooled together and used for ( A ). Similarly, the corresponding Illumina data were pooled together for ( B ). The Illumina molecular counts data were derived using 5′ molecular tags at the RT step as described in material and methods. The total number of molecules presented on the x-axis corresponds to 3.5 pgs of ERCC RNA. In both axes the log10 transformation of the original count number is used.

    Article Snippet: For cDNA quantification, the ONT MinION platform can be used instead of the Illumina platform as it provides comparable quantitative data.

    Techniques: Derivative Assay, Transformation Assay

    Detection of different cDNA species for the RPL41 gene from either the Illumina HiSeq 2500 platform, the ONT MinION platform or the PacBio RS II platform. ONT MinION reads that were sequenced as full length, as defined by the presence of both the 5′ and 3′ RT adaptors, are presented. For the PacBio RS II example the corresponding “Circular Consensus Sequencing” reads are presented. These reads correspond to fully sequenced molecules. mRNA molecules from GenBank for the RPL41 gene are also shown. For the Illumina data a pileup of the sequenced paired-end fragments is presented.

    Journal: Scientific Reports

    Article Title: Benchmarking of the Oxford Nanopore MinION sequencing for quantitative and qualitative assessment of cDNA populations

    doi: 10.1038/srep31602

    Figure Lengend Snippet: Detection of different cDNA species for the RPL41 gene from either the Illumina HiSeq 2500 platform, the ONT MinION platform or the PacBio RS II platform. ONT MinION reads that were sequenced as full length, as defined by the presence of both the 5′ and 3′ RT adaptors, are presented. For the PacBio RS II example the corresponding “Circular Consensus Sequencing” reads are presented. These reads correspond to fully sequenced molecules. mRNA molecules from GenBank for the RPL41 gene are also shown. For the Illumina data a pileup of the sequenced paired-end fragments is presented.

    Article Snippet: For cDNA quantification, the ONT MinION platform can be used instead of the Illumina platform as it provides comparable quantitative data.

    Techniques: Sequencing

    Estimation of the HEK-293 cDNA isoform abundance with three sequencing platforms. The comparison between the cDNA isoform abundance estimated from the Illumina HiSeq 2500 platform and from the PacBio RS II platform is presented in ( A ). The expression level of the HEK-293 isoforms estimated with the ONT MinION platform is compared with the one calculated from either the PacBio RS II ( B ) or the Illumina HiSeq 2500 platform ( C ). For the Illumina HiSeq 2500 platform the expression level, presented as TPM, was estimated with the Sailfish 21 software. For the PacBio RS II or the ONT MinION platform the counts of sequenced molecules per isoform are presented.

    Journal: Scientific Reports

    Article Title: Benchmarking of the Oxford Nanopore MinION sequencing for quantitative and qualitative assessment of cDNA populations

    doi: 10.1038/srep31602

    Figure Lengend Snippet: Estimation of the HEK-293 cDNA isoform abundance with three sequencing platforms. The comparison between the cDNA isoform abundance estimated from the Illumina HiSeq 2500 platform and from the PacBio RS II platform is presented in ( A ). The expression level of the HEK-293 isoforms estimated with the ONT MinION platform is compared with the one calculated from either the PacBio RS II ( B ) or the Illumina HiSeq 2500 platform ( C ). For the Illumina HiSeq 2500 platform the expression level, presented as TPM, was estimated with the Sailfish 21 software. For the PacBio RS II or the ONT MinION platform the counts of sequenced molecules per isoform are presented.

    Article Snippet: For cDNA quantification, the ONT MinION platform can be used instead of the Illumina platform as it provides comparable quantitative data.

    Techniques: Sequencing, Expressing, Software

    Effect of the GC content and ERCC length on the estimation of the ERCC cDNA abundance with the ONT MinION platform. The figures present deviations of the ERCC expression level estimates with the ONT MinION platform from the Ambion RNA molecular counts ( A,C ) or from the Illumina HiSeq 2500/MiSeq estimated cDNA abundance ( B,D ) as a function of the GC content ( A,B ) and the ERCC length ( C,D ). We plot the log2 ratio of observed (ONT MinION) to expected (Ambion, Illumina) read counts for the ERCC spike-ins (y-axis, log) for each of the samples relative to their length or GC content (x-axis). Due to the variable sequencing depth from each ERCC MinION experiment, each point is the average value from different MinION flow cell runs if at least 5 reads have been detected for this point in the corresponding MinION runs. The points are colored differently based on the number of flow cell runs in which they were detected (red, green, cyan, purple correspond to values derived from one, two, three or four flow cell runs respectively). The standard deviation is also presented for points with values from two or more MinION runs.

    Journal: Scientific Reports

    Article Title: Benchmarking of the Oxford Nanopore MinION sequencing for quantitative and qualitative assessment of cDNA populations

    doi: 10.1038/srep31602

    Figure Lengend Snippet: Effect of the GC content and ERCC length on the estimation of the ERCC cDNA abundance with the ONT MinION platform. The figures present deviations of the ERCC expression level estimates with the ONT MinION platform from the Ambion RNA molecular counts ( A,C ) or from the Illumina HiSeq 2500/MiSeq estimated cDNA abundance ( B,D ) as a function of the GC content ( A,B ) and the ERCC length ( C,D ). We plot the log2 ratio of observed (ONT MinION) to expected (Ambion, Illumina) read counts for the ERCC spike-ins (y-axis, log) for each of the samples relative to their length or GC content (x-axis). Due to the variable sequencing depth from each ERCC MinION experiment, each point is the average value from different MinION flow cell runs if at least 5 reads have been detected for this point in the corresponding MinION runs. The points are colored differently based on the number of flow cell runs in which they were detected (red, green, cyan, purple correspond to values derived from one, two, three or four flow cell runs respectively). The standard deviation is also presented for points with values from two or more MinION runs.

    Article Snippet: The abundance of the different ERCC cDNA molecules sequenced from either the Illumina HiSeq 2500/MiSeq platforms, the PacBio RS II platform or the ONT MinION is comparable To test whether the ONT MinION performs equally well as the Illumina platforms in estimating the ERCC cDNA abundance, the same full length ERCC cDNA population used in the ONT MinION experiments, was sequenced on an Illumina HiSeq 2500 or MiSeq instruments.

    Techniques: Expressing, Sequencing, Flow Cytometry, Derivative Assay, Standard Deviation

    Estimation of the ERCC cDNA abundance with the ONT MinION platform. We compared the ERCC cDNA abundance estimated from the ONT MinION ( A ) and the Illumina HiSeq 2500 or MiSeq platforms ( B ) against the expected number of RNA molecules as provided from the manufacturer (Ambion). The template reads from the ERCC MinION experiments number 1, 2, 3, 4 were pooled together and used for ( A ). Similarly, the corresponding Illumina data were pooled together for ( B ). The Illumina molecular counts data were derived using 5′ molecular tags at the RT step as described in material and methods. The total number of molecules presented on the x-axis corresponds to 3.5 pgs of ERCC RNA. In both axes the log10 transformation of the original count number is used.

    Journal: Scientific Reports

    Article Title: Benchmarking of the Oxford Nanopore MinION sequencing for quantitative and qualitative assessment of cDNA populations

    doi: 10.1038/srep31602

    Figure Lengend Snippet: Estimation of the ERCC cDNA abundance with the ONT MinION platform. We compared the ERCC cDNA abundance estimated from the ONT MinION ( A ) and the Illumina HiSeq 2500 or MiSeq platforms ( B ) against the expected number of RNA molecules as provided from the manufacturer (Ambion). The template reads from the ERCC MinION experiments number 1, 2, 3, 4 were pooled together and used for ( A ). Similarly, the corresponding Illumina data were pooled together for ( B ). The Illumina molecular counts data were derived using 5′ molecular tags at the RT step as described in material and methods. The total number of molecules presented on the x-axis corresponds to 3.5 pgs of ERCC RNA. In both axes the log10 transformation of the original count number is used.

    Article Snippet: The abundance of the different ERCC cDNA molecules sequenced from either the Illumina HiSeq 2500/MiSeq platforms, the PacBio RS II platform or the ONT MinION is comparable To test whether the ONT MinION performs equally well as the Illumina platforms in estimating the ERCC cDNA abundance, the same full length ERCC cDNA population used in the ONT MinION experiments, was sequenced on an Illumina HiSeq 2500 or MiSeq instruments.

    Techniques: Derivative Assay, Transformation Assay

    Relative proportion of sRNA candidates in different classes . (a) 454 sequencing: distribution of reads mapped to the S. meliloti 1021 genome and distribution of the analyzed contigs according to the general classification (Figure 3). Left circle diagram: light colored (I) and colored (II), number of reads derived from sample 1 and 2. Reads in sample 1 and 2: non-mapped, 48,159 and 57,964; rRNA genes, 67,891 and 176,848; tRNA genes, 188,121 and 79,789; repeats, 3,029 and 6,206; IGRs or ORFs, 77,326 and 140,702. Right circle diagram: light colored (I), colored (II) and dark colored (I+II) represent the number of RNA candidates derived from sample 1, sample 2, and both samples, respectively: trans-encoded sRNAs, 28, 38, 85; cis-encoded antisense sRNAs, 9, 52, 35; mRNA leader transcripts, 46, 151, 181; sense sRNAs 28, 363, 56; ORFs 0, 4, 4. (b) Illumina/Solexa sequencing: Distribution of reads mapped to the S. meliloti 1021 genome. Reads: non-mapped, 1,179,722; rRNA genes, 3,405,289; tRNA genes, 1,058,534; repeats, 111,355; IGR and ORFs, 711,851. Dark green segment: contigs for 44 putative trans-encoded sRNAs. (c) Microarray-based analysis and (d) Affymetrix Symbiosis Chip-based analysis: distribution of sRNA candidates. Segment numbers represent subtypes. Microarray data: type 1 and 2 trans-encoded sRNAs, 264 and 721 candidates; type 1, 2 and 3 cis-encoded antisense sRNAs, 25, 587 and 59; mRNA leader transcripts, 250. Affymetrix Symbiosis Chip data: type 1 and 2 trans-encoded sRNAs, 60 and 174; type 1, 2 and 3 cis-encoded antisense sRNAs, 3, 4 and 27; mRNA leader, 112.

    Journal: BMC Genomics

    Article Title: A genome-wide survey of sRNAs in the symbiotic nitrogen-fixing alpha-proteobacterium Sinorhizobium meliloti

    doi: 10.1186/1471-2164-11-245

    Figure Lengend Snippet: Relative proportion of sRNA candidates in different classes . (a) 454 sequencing: distribution of reads mapped to the S. meliloti 1021 genome and distribution of the analyzed contigs according to the general classification (Figure 3). Left circle diagram: light colored (I) and colored (II), number of reads derived from sample 1 and 2. Reads in sample 1 and 2: non-mapped, 48,159 and 57,964; rRNA genes, 67,891 and 176,848; tRNA genes, 188,121 and 79,789; repeats, 3,029 and 6,206; IGRs or ORFs, 77,326 and 140,702. Right circle diagram: light colored (I), colored (II) and dark colored (I+II) represent the number of RNA candidates derived from sample 1, sample 2, and both samples, respectively: trans-encoded sRNAs, 28, 38, 85; cis-encoded antisense sRNAs, 9, 52, 35; mRNA leader transcripts, 46, 151, 181; sense sRNAs 28, 363, 56; ORFs 0, 4, 4. (b) Illumina/Solexa sequencing: Distribution of reads mapped to the S. meliloti 1021 genome. Reads: non-mapped, 1,179,722; rRNA genes, 3,405,289; tRNA genes, 1,058,534; repeats, 111,355; IGR and ORFs, 711,851. Dark green segment: contigs for 44 putative trans-encoded sRNAs. (c) Microarray-based analysis and (d) Affymetrix Symbiosis Chip-based analysis: distribution of sRNA candidates. Segment numbers represent subtypes. Microarray data: type 1 and 2 trans-encoded sRNAs, 264 and 721 candidates; type 1, 2 and 3 cis-encoded antisense sRNAs, 25, 587 and 59; mRNA leader transcripts, 250. Affymetrix Symbiosis Chip data: type 1 and 2 trans-encoded sRNAs, 60 and 174; type 1, 2 and 3 cis-encoded antisense sRNAs, 3, 4 and 27; mRNA leader, 112.

    Article Snippet: This table lists the sRNAs identified by 454 and SolexA/Illumina cDNA sequencing in this study.

    Techniques: Sequencing, Derivative Assay, Microarray, Chromatin Immunoprecipitation

    Venn diagram comparing trans-encoded sRNA candidates identified by 454 sequencing, Illumina/Solexa sequencing, and microarray hybridizations . In some cases a sRNA region detected by one method overlaps with multiple regions detected by one or both of the other methods. This is indictated by the colors of the numbers in the fields representing the overlaps. Numbers in brackets indicate discrepancies in the classification of sRNA regions identified by different methods. Small numbers indicate 454 deep sequencing candidates not classified as trans-encoded sRNA.

    Journal: BMC Genomics

    Article Title: A genome-wide survey of sRNAs in the symbiotic nitrogen-fixing alpha-proteobacterium Sinorhizobium meliloti

    doi: 10.1186/1471-2164-11-245

    Figure Lengend Snippet: Venn diagram comparing trans-encoded sRNA candidates identified by 454 sequencing, Illumina/Solexa sequencing, and microarray hybridizations . In some cases a sRNA region detected by one method overlaps with multiple regions detected by one or both of the other methods. This is indictated by the colors of the numbers in the fields representing the overlaps. Numbers in brackets indicate discrepancies in the classification of sRNA regions identified by different methods. Small numbers indicate 454 deep sequencing candidates not classified as trans-encoded sRNA.

    Article Snippet: This table lists the sRNAs identified by 454 and SolexA/Illumina cDNA sequencing in this study.

    Techniques: Sequencing, Microarray

    Expression pattern of sequenced non-coding transcripts . Expression pattern of ( a ) trans-encoded sRNA, ( b ) cis-encoded antisense sRNAs and ( c ) mRNA leader transcripts identified by deep sequencing: log, stat, heat, cold, acidic, basic, oxidative represent the analyzed stress conditions. Grey, white and green boxes indicate no signal, weak signal (less than 8-fold) and strong signal (≥ 8-fold), respectively. * indicates candidates uniquely identified with Illumina/Solexa sequencing.

    Journal: BMC Genomics

    Article Title: A genome-wide survey of sRNAs in the symbiotic nitrogen-fixing alpha-proteobacterium Sinorhizobium meliloti

    doi: 10.1186/1471-2164-11-245

    Figure Lengend Snippet: Expression pattern of sequenced non-coding transcripts . Expression pattern of ( a ) trans-encoded sRNA, ( b ) cis-encoded antisense sRNAs and ( c ) mRNA leader transcripts identified by deep sequencing: log, stat, heat, cold, acidic, basic, oxidative represent the analyzed stress conditions. Grey, white and green boxes indicate no signal, weak signal (less than 8-fold) and strong signal (≥ 8-fold), respectively. * indicates candidates uniquely identified with Illumina/Solexa sequencing.

    Article Snippet: This table lists the sRNAs identified by 454 and SolexA/Illumina cDNA sequencing in this study.

    Techniques: Expressing, Sequencing

    Viral RNA fragments produced by RNase L and RNase A. HCV and PV RNAs were incubated with RNase L and RNase A to produce RNA fragments for 2′, 3′-cyclic phosphate cDNA synthesis and sequencing. Agarose gel electrophoresis and ethidium bromide staining revealed the size of viral RNA fragments. ( A ) Diagram of HCV and PV RNAs. HCV RNA is 9648 bases long. PV RNA is 7500 bases long. ( B ) Viral RNAs incubated with RNase L. HCV and PV RNAs were incubated with RNase L for 20 min in the absence of 2-5A (no 2-5A), or with RNase L and 2-5A for 0, 2.5, 5, 10 and 20 min. ( C ) Viral RNAs incubated with RNase A. HCV and PV RNAs were incubated for 20 min in the absence of RNase A (−), and the presence of RNase A for 0, 2.5, 5, 10 and 20 min.

    Journal: Nucleic Acids Research

    Article Title: Ribonuclease L and metal-ion-independent endoribonuclease cleavage sites in host and viral RNAs

    doi: 10.1093/nar/gku118

    Figure Lengend Snippet: Viral RNA fragments produced by RNase L and RNase A. HCV and PV RNAs were incubated with RNase L and RNase A to produce RNA fragments for 2′, 3′-cyclic phosphate cDNA synthesis and sequencing. Agarose gel electrophoresis and ethidium bromide staining revealed the size of viral RNA fragments. ( A ) Diagram of HCV and PV RNAs. HCV RNA is 9648 bases long. PV RNA is 7500 bases long. ( B ) Viral RNAs incubated with RNase L. HCV and PV RNAs were incubated with RNase L for 20 min in the absence of 2-5A (no 2-5A), or with RNase L and 2-5A for 0, 2.5, 5, 10 and 20 min. ( C ) Viral RNAs incubated with RNase A. HCV and PV RNAs were incubated for 20 min in the absence of RNase A (−), and the presence of RNase A for 0, 2.5, 5, 10 and 20 min.

    Article Snippet: We optimized and validated 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing methods using viral RNAs cleaved with purified RNase L, viral RNAs cleaved with purified RNase A and RNA from uninfected and poliovirus-infected HeLa cells.

    Techniques: Produced, Incubation, Sequencing, Agarose Gel Electrophoresis, Staining

    Endoribonuclease cleavage sites in PV RNA isolated from HeLa cells. RNAs from PV-infected HeLa cells ( Figure 3 B) were used for 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing. ( A ) Location and frequency of cleavage sites in PV RNA from W12 HeLa cells. X-axis: Nucleotide position in PV RNA. Y-axis: Number of distinct UMI-tagged linkers detected at each cleavage site. ( B ) Dinucleotide specificity of cleavage sites in PV RNA from W12 HeLa cells. X-axis: Dinucleotide at the 3′-end of PV RNA fragments (adjacent to 8 base UMI sequence in RNA linkers as illustrated in Supplementary Figure S1 ). Y-axis: Percent of PV cDNA reads. ( C ) Location and frequency of cleavage sites in PV RNA from M25 HeLa cells. X-axis: Nucleotide position in PV RNA. Y-axis: Number of distinct UMI-tagged linkers detected at each cleavage site. ( D ) Dinucleotide specificity of cleavage sites in PV RNA from M25 HeLa cells. X-axis: Dinucleotide at the 3′-end of PV RNA fragments. Y-axis: Percent of PV cDNA reads.

    Journal: Nucleic Acids Research

    Article Title: Ribonuclease L and metal-ion-independent endoribonuclease cleavage sites in host and viral RNAs

    doi: 10.1093/nar/gku118

    Figure Lengend Snippet: Endoribonuclease cleavage sites in PV RNA isolated from HeLa cells. RNAs from PV-infected HeLa cells ( Figure 3 B) were used for 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing. ( A ) Location and frequency of cleavage sites in PV RNA from W12 HeLa cells. X-axis: Nucleotide position in PV RNA. Y-axis: Number of distinct UMI-tagged linkers detected at each cleavage site. ( B ) Dinucleotide specificity of cleavage sites in PV RNA from W12 HeLa cells. X-axis: Dinucleotide at the 3′-end of PV RNA fragments (adjacent to 8 base UMI sequence in RNA linkers as illustrated in Supplementary Figure S1 ). Y-axis: Percent of PV cDNA reads. ( C ) Location and frequency of cleavage sites in PV RNA from M25 HeLa cells. X-axis: Nucleotide position in PV RNA. Y-axis: Number of distinct UMI-tagged linkers detected at each cleavage site. ( D ) Dinucleotide specificity of cleavage sites in PV RNA from M25 HeLa cells. X-axis: Dinucleotide at the 3′-end of PV RNA fragments. Y-axis: Percent of PV cDNA reads.

    Article Snippet: We optimized and validated 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing methods using viral RNAs cleaved with purified RNase L, viral RNAs cleaved with purified RNase A and RNA from uninfected and poliovirus-infected HeLa cells.

    Techniques: Isolation, Infection, Sequencing

    Endoribonuclease cleavage sites in rRNAs from M25 HeLa cells. RNAs from mock-infected and PV-infected M25 HeLa cells ( Figure 3 B) were used for 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing. The location and frequency of cleavage sites in 28S rRNA ( A ), 18S rRNA ( B ), 5.8S rRNA ( C ) and 5S rRNA ( D ) are shown for mock-infected and PV-infected RNA samples isolated at 8 hpa. X-axis: Nucleotide position of each RNA. Y-axis: Percentage of total UMIs at each cleavage site. Dinucleotides at the 3′-end of abundant RNA fragments are annotated at the corresponding positions in the graphs. The locations of GC-rich expansion segments are highlighted by light blue rectangles.

    Journal: Nucleic Acids Research

    Article Title: Ribonuclease L and metal-ion-independent endoribonuclease cleavage sites in host and viral RNAs

    doi: 10.1093/nar/gku118

    Figure Lengend Snippet: Endoribonuclease cleavage sites in rRNAs from M25 HeLa cells. RNAs from mock-infected and PV-infected M25 HeLa cells ( Figure 3 B) were used for 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing. The location and frequency of cleavage sites in 28S rRNA ( A ), 18S rRNA ( B ), 5.8S rRNA ( C ) and 5S rRNA ( D ) are shown for mock-infected and PV-infected RNA samples isolated at 8 hpa. X-axis: Nucleotide position of each RNA. Y-axis: Percentage of total UMIs at each cleavage site. Dinucleotides at the 3′-end of abundant RNA fragments are annotated at the corresponding positions in the graphs. The locations of GC-rich expansion segments are highlighted by light blue rectangles.

    Article Snippet: We optimized and validated 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing methods using viral RNAs cleaved with purified RNase L, viral RNAs cleaved with purified RNase A and RNA from uninfected and poliovirus-infected HeLa cells.

    Techniques: Infection, Sequencing, Isolation

    Frequency, location and dinucleotide specificity of cleavage sites in viral RNAs. 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing was used to analyze the viral RNA fragments shown in Figure 1 . ( A ) Frequency, location and dinucleotide specificity of endoribonuclease cleavage sites in HCV RNA (from 20 min samples). ( B ) Frequency, location and dinucleotide specificity of cleavage sites in PV RNA (from 20 min samples).

    Journal: Nucleic Acids Research

    Article Title: Ribonuclease L and metal-ion-independent endoribonuclease cleavage sites in host and viral RNAs

    doi: 10.1093/nar/gku118

    Figure Lengend Snippet: Frequency, location and dinucleotide specificity of cleavage sites in viral RNAs. 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing was used to analyze the viral RNA fragments shown in Figure 1 . ( A ) Frequency, location and dinucleotide specificity of endoribonuclease cleavage sites in HCV RNA (from 20 min samples). ( B ) Frequency, location and dinucleotide specificity of cleavage sites in PV RNA (from 20 min samples).

    Article Snippet: We optimized and validated 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing methods using viral RNAs cleaved with purified RNase L, viral RNAs cleaved with purified RNase A and RNA from uninfected and poliovirus-infected HeLa cells.

    Techniques: Sequencing

    Endoribonuclease cleavage sites in rRNAs from W12 HeLa cells. RNAs from mock-infected and PV-infected W12 HeLa cells ( Figure 3 B) were used for 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing. The location and frequency of cleavage sites in 28S rRNA ( A ), 18S rRNA ( B ), 5.8S rRNA ( C ) and 5S rRNA ( D ) are shown for mock-infected and PV-infected RNA samples isolated at 8 hpa. X-axis: Nucleotide position of each RNA. Y-axis: Percentage of total UMIs at each cleavage site. Dinucleotides at the 3′-end of abundant RNA fragments are annotated at the corresponding positions in the graphs. The locations of GC-rich expansion segments are highlighted by light blue rectangles. RNase L cleavage sites are highlighted in red.

    Journal: Nucleic Acids Research

    Article Title: Ribonuclease L and metal-ion-independent endoribonuclease cleavage sites in host and viral RNAs

    doi: 10.1093/nar/gku118

    Figure Lengend Snippet: Endoribonuclease cleavage sites in rRNAs from W12 HeLa cells. RNAs from mock-infected and PV-infected W12 HeLa cells ( Figure 3 B) were used for 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing. The location and frequency of cleavage sites in 28S rRNA ( A ), 18S rRNA ( B ), 5.8S rRNA ( C ) and 5S rRNA ( D ) are shown for mock-infected and PV-infected RNA samples isolated at 8 hpa. X-axis: Nucleotide position of each RNA. Y-axis: Percentage of total UMIs at each cleavage site. Dinucleotides at the 3′-end of abundant RNA fragments are annotated at the corresponding positions in the graphs. The locations of GC-rich expansion segments are highlighted by light blue rectangles. RNase L cleavage sites are highlighted in red.

    Article Snippet: We optimized and validated 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing methods using viral RNAs cleaved with purified RNase L, viral RNAs cleaved with purified RNase A and RNA from uninfected and poliovirus-infected HeLa cells.

    Techniques: Infection, Sequencing, Isolation

    Host and viral RNA from mock-infected and PV-infected HeLa cells. RNA was isolated from mock-infected and PV-infected HeLa cells for 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing. ( A ) PV infection. W12 and M25 HeLa cells were infected with PV using 10 PFUs per cell. PV titers determined by plaque assay and plotted versus time (hpa). ( B ) RNA from PV-infected HeLa cells. RNA was isolated from infected cells, fractionated by agarose gel electrophoresis and visualized using ethidium bromide and UV light. ( C and D ) cDNA reads from W12 (C) and M25 (D) HeLa cells. The RNAs shown in Figure 3 B were used for 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing. Amounts of host and viral cDNA in each sample are plotted (data from Supplementary Table S3 ).

    Journal: Nucleic Acids Research

    Article Title: Ribonuclease L and metal-ion-independent endoribonuclease cleavage sites in host and viral RNAs

    doi: 10.1093/nar/gku118

    Figure Lengend Snippet: Host and viral RNA from mock-infected and PV-infected HeLa cells. RNA was isolated from mock-infected and PV-infected HeLa cells for 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing. ( A ) PV infection. W12 and M25 HeLa cells were infected with PV using 10 PFUs per cell. PV titers determined by plaque assay and plotted versus time (hpa). ( B ) RNA from PV-infected HeLa cells. RNA was isolated from infected cells, fractionated by agarose gel electrophoresis and visualized using ethidium bromide and UV light. ( C and D ) cDNA reads from W12 (C) and M25 (D) HeLa cells. The RNAs shown in Figure 3 B were used for 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing. Amounts of host and viral cDNA in each sample are plotted (data from Supplementary Table S3 ).

    Article Snippet: We optimized and validated 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing methods using viral RNAs cleaved with purified RNase L, viral RNAs cleaved with purified RNase A and RNA from uninfected and poliovirus-infected HeLa cells.

    Techniques: Infection, Isolation, Sequencing, Plaque Assay, Agarose Gel Electrophoresis