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  • 99
    Illumina Inc truseq rna sample preparation kit
    Improved comparability across degradation effects and library preparation methods. ( a ) Correlation of unnormalized reads from normally processed iPSC <t>RNA</t> (HICL) and in-process degraded iPSC RNA (HICL_D). Reads for 51,552 loci over the read cutoff of 40 were plotted. ( b ) Correlation of SNV-normalized reads from the HICL and HICL_D samples. ( c ) Agreement of estimates for gene expression in HICL and HICL_D samples estimated by unnormalized reads and SNV-normalized reads. ( d ) Correlation of unnormalized reads generated by <t>TruSeq</t> method (HICL) and NexTera method (HICL_N). Reads for 53,585 loci were plotted. ( e ) Correlation of SNV-normalized reads from the HICL and HICL_N samples. ( f ) Agreement of estimates for gene expression in HICL and HICL_N samples estimated by unnormalized reads and SNV-normalized reads.
    Truseq Rna Sample Preparation Kit, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 10537 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Illumina Inc truseq rna
    Experimental pipeline of SUPeR-seq, and its sensitivity at the whole-transcriptome scale. a The schematic of SUPeR-seq analysis. A single cell is lysed to release RNAs. RNAs are then reverse transcribed into first-strand cDNAs using random primers with a fixed anchor sequence (AnchorX-T 15 N 6 ). Unreacted primers are then digested using ExoSAP-IT, followed by adding poly(A) tails to the 3′ ends of the first-strand cDNAs using dATP doped with 1 % ddATP to restrict the length of poly(A) tails. Second-strand cDNAs are synthesized using poly(T) primers with a different anchor sequence (AnchorY-T 24 ). Then the double-stranded cDNAs are evenly amplified by PCR using AnchorX-T 15 and AnchorY-T 24 primers. Finally the purified single cell cDNAs are used to prepare sequencing libraries following Illumina’s <t>TruSeq</t> DNA sample preparation protocols. b Detection sensitivity of SUPeR-seq on poly(A)- genes in individual cells. We identified 696 poly(A)- genes by bulk <t>RNA</t> sequencing, of which around 30 % could be recovered in a single cell by SUPeR-seq (for details, see Additional file 3 ). When merged the SUPeR-seq data of the seven single cell samples together, over 50 % of these 696 genes could be successfully recovered by SUPeR-seq in at least one cell. c The number of genes detected from individual and bulk HEK293T cells using different protocols. SUPeR-seq detected 10,911 genes on average from an individual cell with FPKM ≥1(Fragments Per Kilobase of exon model per Million mapped reads), 19.3 % more than the Tang2009 protocol did (9148 genes on average). For comparison, 14,931 genes were detected with FPKM ≥1 in the four rRNA-depleted total RNA samples from bulk HEK293T cells, and 15,535 genes were detected in the four oligo(dT)-enriched total RNA samples from bulk HEK293T cells
    Truseq Rna, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 7061 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 7061 article reviews
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    99
    Illumina Inc truseq rna sample prep kit
    Experimental pipeline of SUPeR-seq, and its sensitivity at the whole-transcriptome scale. a The schematic of SUPeR-seq analysis. A single cell is lysed to release RNAs. RNAs are then reverse transcribed into first-strand cDNAs using random primers with a fixed anchor sequence (AnchorX-T 15 N 6 ). Unreacted primers are then digested using ExoSAP-IT, followed by adding poly(A) tails to the 3′ ends of the first-strand cDNAs using dATP doped with 1 % ddATP to restrict the length of poly(A) tails. Second-strand cDNAs are synthesized using poly(T) primers with a different anchor sequence (AnchorY-T 24 ). Then the double-stranded cDNAs are evenly amplified by PCR using AnchorX-T 15 and AnchorY-T 24 primers. Finally the purified single cell cDNAs are used to prepare sequencing libraries following Illumina’s <t>TruSeq</t> DNA sample preparation protocols. b Detection sensitivity of SUPeR-seq on poly(A)- genes in individual cells. We identified 696 poly(A)- genes by bulk <t>RNA</t> sequencing, of which around 30 % could be recovered in a single cell by SUPeR-seq (for details, see Additional file 3 ). When merged the SUPeR-seq data of the seven single cell samples together, over 50 % of these 696 genes could be successfully recovered by SUPeR-seq in at least one cell. c The number of genes detected from individual and bulk HEK293T cells using different protocols. SUPeR-seq detected 10,911 genes on average from an individual cell with FPKM ≥1(Fragments Per Kilobase of exon model per Million mapped reads), 19.3 % more than the Tang2009 protocol did (9148 genes on average). For comparison, 14,931 genes were detected with FPKM ≥1 in the four rRNA-depleted total RNA samples from bulk HEK293T cells, and 15,535 genes were detected in the four oligo(dT)-enriched total RNA samples from bulk HEK293T cells
    Truseq Rna Sample Prep Kit, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 7813 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 7813 article reviews
    Price from $9.99 to $1999.99
    truseq rna sample prep kit - by Bioz Stars, 2020-09
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    94
    Illumina Inc truseq rna sample prep kit v2
    Experimental pipeline of SUPeR-seq, and its sensitivity at the whole-transcriptome scale. a The schematic of SUPeR-seq analysis. A single cell is lysed to release RNAs. RNAs are then reverse transcribed into first-strand cDNAs using random primers with a fixed anchor sequence (AnchorX-T 15 N 6 ). Unreacted primers are then digested using ExoSAP-IT, followed by adding poly(A) tails to the 3′ ends of the first-strand cDNAs using dATP doped with 1 % ddATP to restrict the length of poly(A) tails. Second-strand cDNAs are synthesized using poly(T) primers with a different anchor sequence (AnchorY-T 24 ). Then the double-stranded cDNAs are evenly amplified by PCR using AnchorX-T 15 and AnchorY-T 24 primers. Finally the purified single cell cDNAs are used to prepare sequencing libraries following Illumina’s <t>TruSeq</t> DNA sample preparation protocols. b Detection sensitivity of SUPeR-seq on poly(A)- genes in individual cells. We identified 696 poly(A)- genes by bulk <t>RNA</t> sequencing, of which around 30 % could be recovered in a single cell by SUPeR-seq (for details, see Additional file 3 ). When merged the SUPeR-seq data of the seven single cell samples together, over 50 % of these 696 genes could be successfully recovered by SUPeR-seq in at least one cell. c The number of genes detected from individual and bulk HEK293T cells using different protocols. SUPeR-seq detected 10,911 genes on average from an individual cell with FPKM ≥1(Fragments Per Kilobase of exon model per Million mapped reads), 19.3 % more than the Tang2009 protocol did (9148 genes on average). For comparison, 14,931 genes were detected with FPKM ≥1 in the four rRNA-depleted total RNA samples from bulk HEK293T cells, and 15,535 genes were detected in the four oligo(dT)-enriched total RNA samples from bulk HEK293T cells
    Truseq Rna Sample Prep Kit V2, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 3517 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Illumina Inc truseq stranded mrna sample preparation kit
    Experimental pipeline of SUPeR-seq, and its sensitivity at the whole-transcriptome scale. a The schematic of SUPeR-seq analysis. A single cell is lysed to release RNAs. RNAs are then reverse transcribed into first-strand cDNAs using random primers with a fixed anchor sequence (AnchorX-T 15 N 6 ). Unreacted primers are then digested using ExoSAP-IT, followed by adding poly(A) tails to the 3′ ends of the first-strand cDNAs using dATP doped with 1 % ddATP to restrict the length of poly(A) tails. Second-strand cDNAs are synthesized using poly(T) primers with a different anchor sequence (AnchorY-T 24 ). Then the double-stranded cDNAs are evenly amplified by PCR using AnchorX-T 15 and AnchorY-T 24 primers. Finally the purified single cell cDNAs are used to prepare sequencing libraries following Illumina’s <t>TruSeq</t> DNA sample preparation protocols. b Detection sensitivity of SUPeR-seq on poly(A)- genes in individual cells. We identified 696 poly(A)- genes by bulk <t>RNA</t> sequencing, of which around 30 % could be recovered in a single cell by SUPeR-seq (for details, see Additional file 3 ). When merged the SUPeR-seq data of the seven single cell samples together, over 50 % of these 696 genes could be successfully recovered by SUPeR-seq in at least one cell. c The number of genes detected from individual and bulk HEK293T cells using different protocols. SUPeR-seq detected 10,911 genes on average from an individual cell with FPKM ≥1(Fragments Per Kilobase of exon model per Million mapped reads), 19.3 % more than the Tang2009 protocol did (9148 genes on average). For comparison, 14,931 genes were detected with FPKM ≥1 in the four rRNA-depleted total RNA samples from bulk HEK293T cells, and 15,535 genes were detected in the four oligo(dT)-enriched total RNA samples from bulk HEK293T cells
    Truseq Stranded Mrna Sample Preparation Kit, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 1819 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Illumina Inc truseq stranded mrna
    RNA-seq profiling of individual kdm2aa -deficient embryos and their siblings. (A) Summary of experimental design. <t>TruSeq</t> stranded <t>mRNA</t> sequencing libraries were prepared from four individual embryos from each genotype at both 5 d.p.f. and 12 d.p.f., totalling 48 libraries. Paired end sequencing with a read length of 75 bp was performed on four lanes of Illumina HiSeq 2500 machines. Sequence was aligned to the GRCz10 reference genome with TopHat and read counts were obtained with HTseq-count. Quality control was performed using QoRTs and outliers removed. 32,261 genes were detected. DESeq2 was used to determine DE genes in each individual clutch, DE genes at each stage by combining the two clutches for each stage whilst controlling for clutch in the DESeq2 model, and also in a combined analysis combining all 4 clutches whilst controlling for stage and clutch. (B) Venn diagram showing the overlap in DE genes between the two 5 d.p.f. experiments and the combined 5 d.p.f. analysis. The increase in power due to the increased sample size enabled an additional 1106 DE genes to be detected. (C) Venn diagram displaying the overlap in DE genes between the two 12 d.p.f. experiments and the combined 12 d.p.f. analysis. An additional 1102 DE genes were detected in the combined analysis. (D) Volcano plot of all detected genes in the combined RNA-seq analysis with the 3752 DE genes shown in red and kdm2aa circled. (E) Box plot of normalised counts for the chromatin modifier nsd1b , with adjusted p-values for individual experiments, stage-specific and combined analysis as indicated by horizontal bars. Data for heterozygous and wild-type siblings are combined. In the figure legend ‘s’ denotes siblings and ‘m’ homozygous mutants. (F) Box plot of normalised counts for dnmt3bb . 2 , a de novo DNA methyl transferase, with adjusted p-values for individual experiments, stage-specific and combined analysis as indicated by horizontal bars. Data for heterozygous and wild-type siblings are combined. In the figure legend ‘s’ denotes siblings and ‘m’ homozygous mutants. (G) Table of enriched Gene Ontology (GO) terms of the biological process (BP) GO domain which overlap in both 5 d.p.f. experiments and also the 5 d.p.f. combined analysis. P-values shown are for the combined analysis. (H) Table of enriched GO terms that overlap in both individual 12 d.p.f. experiments and the 12 d.p.f. combined analysis, with p-values for the combined analysis shown.
    Truseq Stranded Mrna, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 2681 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Illumina Inc truseq small rna sample prep kit
    Comparative schematic of small <t>RNA</t> barcoding methods. The three methods start with ligation of a 3' and 5' RNA adapter to generate a substrate for RT-PCR. In the pre-PCR barcoding method, the barcode is incorporated in the 5' adapter. In the <t>TruSeq</t> method, the barcode is incorporated in one of the RT-PCR primers. In the PALM barcoding method, the amplified RT-PCR product is A-tailed and ligated to a T-tailed barcoded adapter.
    Truseq Small Rna Sample Prep Kit, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 2056 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Illumina Inc truseq stranded total rna sample preparation kit
    Polyomavirus genome and work flow. (A) Polyomavirus genome including origin and polyadenylation sites. Early gene splicing shown in blue. Late gene splicing shown in red. (B) Schematic of read-through of the polyadenylation site during the late phase of infection. Late transcripts must read through the entire viral genome at least once to allow for the late leader exon (L) to splice properly. This results in spliced late mRNAs with at least two tandem repeats of the late leader exon. (C) Work flow of experiments. NIH 3T6 cells were infected with the Py59RA strain of polyomavirus and either harvested at different time points or treated with aphidicolin to block DNA replication and keep the infection in the early phase for 48 hours. Total <t>RNA</t> was collected and used to synthesize stranded cDNA libraries using the Illumina <t>TruSeq</t> Stranded Total RNA Preparation kit. Samples were run on the Hiseq 2000 sequencer and aligned to both the Py59RA and mouse host genomes.
    Truseq Stranded Total Rna Sample Preparation Kit, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 1202 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/truseq stranded total rna sample preparation kit/product/Illumina Inc
    Average 99 stars, based on 1202 article reviews
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    99
    Illumina Inc truseq stranded total rna sample prep kit
    Polyomavirus genome and work flow. (A) Polyomavirus genome including origin and polyadenylation sites. Early gene splicing shown in blue. Late gene splicing shown in red. (B) Schematic of read-through of the polyadenylation site during the late phase of infection. Late transcripts must read through the entire viral genome at least once to allow for the late leader exon (L) to splice properly. This results in spliced late mRNAs with at least two tandem repeats of the late leader exon. (C) Work flow of experiments. NIH 3T6 cells were infected with the Py59RA strain of polyomavirus and either harvested at different time points or treated with aphidicolin to block DNA replication and keep the infection in the early phase for 48 hours. Total <t>RNA</t> was collected and used to synthesize stranded cDNA libraries using the Illumina <t>TruSeq</t> Stranded Total RNA Preparation kit. Samples were run on the Hiseq 2000 sequencer and aligned to both the Py59RA and mouse host genomes.
    Truseq Stranded Total Rna Sample Prep Kit, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 953 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/truseq stranded total rna sample prep kit/product/Illumina Inc
    Average 99 stars, based on 953 article reviews
    Price from $9.99 to $1999.99
    truseq stranded total rna sample prep kit - by Bioz Stars, 2020-09
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    99
    Illumina Inc truseq stranded total rna
    Polyomavirus genome and work flow. (A) Polyomavirus genome including origin and polyadenylation sites. Early gene splicing shown in blue. Late gene splicing shown in red. (B) Schematic of read-through of the polyadenylation site during the late phase of infection. Late transcripts must read through the entire viral genome at least once to allow for the late leader exon (L) to splice properly. This results in spliced late mRNAs with at least two tandem repeats of the late leader exon. (C) Work flow of experiments. NIH 3T6 cells were infected with the Py59RA strain of polyomavirus and either harvested at different time points or treated with aphidicolin to block DNA replication and keep the infection in the early phase for 48 hours. Total <t>RNA</t> was collected and used to synthesize stranded cDNA libraries using the Illumina <t>TruSeq</t> Stranded Total RNA Preparation kit. Samples were run on the Hiseq 2000 sequencer and aligned to both the Py59RA and mouse host genomes.
    Truseq Stranded Total Rna, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 1751 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/truseq stranded total rna/product/Illumina Inc
    Average 99 stars, based on 1751 article reviews
    Price from $9.99 to $1999.99
    truseq stranded total rna - by Bioz Stars, 2020-09
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    Image Search Results


    Improved comparability across degradation effects and library preparation methods. ( a ) Correlation of unnormalized reads from normally processed iPSC RNA (HICL) and in-process degraded iPSC RNA (HICL_D). Reads for 51,552 loci over the read cutoff of 40 were plotted. ( b ) Correlation of SNV-normalized reads from the HICL and HICL_D samples. ( c ) Agreement of estimates for gene expression in HICL and HICL_D samples estimated by unnormalized reads and SNV-normalized reads. ( d ) Correlation of unnormalized reads generated by TruSeq method (HICL) and NexTera method (HICL_N). Reads for 53,585 loci were plotted. ( e ) Correlation of SNV-normalized reads from the HICL and HICL_N samples. ( f ) Agreement of estimates for gene expression in HICL and HICL_N samples estimated by unnormalized reads and SNV-normalized reads.

    Journal: Scientific Reports

    Article Title: Normalization of human RNA-seq experiments using chimpanzee RNA as a spike-in standard

    doi: 10.1038/srep31923

    Figure Lengend Snippet: Improved comparability across degradation effects and library preparation methods. ( a ) Correlation of unnormalized reads from normally processed iPSC RNA (HICL) and in-process degraded iPSC RNA (HICL_D). Reads for 51,552 loci over the read cutoff of 40 were plotted. ( b ) Correlation of SNV-normalized reads from the HICL and HICL_D samples. ( c ) Agreement of estimates for gene expression in HICL and HICL_D samples estimated by unnormalized reads and SNV-normalized reads. ( d ) Correlation of unnormalized reads generated by TruSeq method (HICL) and NexTera method (HICL_N). Reads for 53,585 loci were plotted. ( e ) Correlation of SNV-normalized reads from the HICL and HICL_N samples. ( f ) Agreement of estimates for gene expression in HICL and HICL_N samples estimated by unnormalized reads and SNV-normalized reads.

    Article Snippet: After second strand synthesis using the second strand master mix in the TruSeq RNA Sample Preparation Kit, cDNA was subjected to the library preparation procedure using NexTera DNA Sample Preparation Kit (Illumina).

    Techniques: Expressing, Generated

    Identification of the BCR - ABL and ABL - BCR fusions in chemically degraded RNA from a KU812 cell line using the TruSeq RNA-seq protocol. a Supporting reads per million reads for BCR - ABL and ABL - BCR at different RIN values (10, 7, 5, 3). b Diagram of ABL and BCR genes and the BCR - ABL and ABL - BCR fusion products showing the different distances of each fusion product from the 3′ end. c Coverage level per million reads as a function of the distance from the 3′ end for the ABL - BCR fusion at different levels of degradation. A loess trend line is depicted for each sample. d Coverage level per million reads as a function of the distance from the 3′ end for the BCR - ABL fusion at different levels of degradation. A loess trend line is depicted for each sample

    Journal: BMC Genomics

    Article Title: Impact of RNA degradation on fusion detection by RNA-seq

    doi: 10.1186/s12864-016-3161-9

    Figure Lengend Snippet: Identification of the BCR - ABL and ABL - BCR fusions in chemically degraded RNA from a KU812 cell line using the TruSeq RNA-seq protocol. a Supporting reads per million reads for BCR - ABL and ABL - BCR at different RIN values (10, 7, 5, 3). b Diagram of ABL and BCR genes and the BCR - ABL and ABL - BCR fusion products showing the different distances of each fusion product from the 3′ end. c Coverage level per million reads as a function of the distance from the 3′ end for the ABL - BCR fusion at different levels of degradation. A loess trend line is depicted for each sample. d Coverage level per million reads as a function of the distance from the 3′ end for the BCR - ABL fusion at different levels of degradation. A loess trend line is depicted for each sample

    Article Snippet: The TruSeq® RNA Sample Preparation v2 Kit (Illumina, San Diego, CA) was used for isolation of polyadenylated mRNA with oligo-dT beads, second strand cDNA synthesis and NGS library preparation.

    Techniques: RNA Sequencing Assay

    Experimental pipeline of SUPeR-seq, and its sensitivity at the whole-transcriptome scale. a The schematic of SUPeR-seq analysis. A single cell is lysed to release RNAs. RNAs are then reverse transcribed into first-strand cDNAs using random primers with a fixed anchor sequence (AnchorX-T 15 N 6 ). Unreacted primers are then digested using ExoSAP-IT, followed by adding poly(A) tails to the 3′ ends of the first-strand cDNAs using dATP doped with 1 % ddATP to restrict the length of poly(A) tails. Second-strand cDNAs are synthesized using poly(T) primers with a different anchor sequence (AnchorY-T 24 ). Then the double-stranded cDNAs are evenly amplified by PCR using AnchorX-T 15 and AnchorY-T 24 primers. Finally the purified single cell cDNAs are used to prepare sequencing libraries following Illumina’s TruSeq DNA sample preparation protocols. b Detection sensitivity of SUPeR-seq on poly(A)- genes in individual cells. We identified 696 poly(A)- genes by bulk RNA sequencing, of which around 30 % could be recovered in a single cell by SUPeR-seq (for details, see Additional file 3 ). When merged the SUPeR-seq data of the seven single cell samples together, over 50 % of these 696 genes could be successfully recovered by SUPeR-seq in at least one cell. c The number of genes detected from individual and bulk HEK293T cells using different protocols. SUPeR-seq detected 10,911 genes on average from an individual cell with FPKM ≥1(Fragments Per Kilobase of exon model per Million mapped reads), 19.3 % more than the Tang2009 protocol did (9148 genes on average). For comparison, 14,931 genes were detected with FPKM ≥1 in the four rRNA-depleted total RNA samples from bulk HEK293T cells, and 15,535 genes were detected in the four oligo(dT)-enriched total RNA samples from bulk HEK293T cells

    Journal: Genome Biology

    Article Title: Single-cell RNA-seq transcriptome analysis of linear and circular RNAs in mouse preimplantation embryos

    doi: 10.1186/s13059-015-0706-1

    Figure Lengend Snippet: Experimental pipeline of SUPeR-seq, and its sensitivity at the whole-transcriptome scale. a The schematic of SUPeR-seq analysis. A single cell is lysed to release RNAs. RNAs are then reverse transcribed into first-strand cDNAs using random primers with a fixed anchor sequence (AnchorX-T 15 N 6 ). Unreacted primers are then digested using ExoSAP-IT, followed by adding poly(A) tails to the 3′ ends of the first-strand cDNAs using dATP doped with 1 % ddATP to restrict the length of poly(A) tails. Second-strand cDNAs are synthesized using poly(T) primers with a different anchor sequence (AnchorY-T 24 ). Then the double-stranded cDNAs are evenly amplified by PCR using AnchorX-T 15 and AnchorY-T 24 primers. Finally the purified single cell cDNAs are used to prepare sequencing libraries following Illumina’s TruSeq DNA sample preparation protocols. b Detection sensitivity of SUPeR-seq on poly(A)- genes in individual cells. We identified 696 poly(A)- genes by bulk RNA sequencing, of which around 30 % could be recovered in a single cell by SUPeR-seq (for details, see Additional file 3 ). When merged the SUPeR-seq data of the seven single cell samples together, over 50 % of these 696 genes could be successfully recovered by SUPeR-seq in at least one cell. c The number of genes detected from individual and bulk HEK293T cells using different protocols. SUPeR-seq detected 10,911 genes on average from an individual cell with FPKM ≥1(Fragments Per Kilobase of exon model per Million mapped reads), 19.3 % more than the Tang2009 protocol did (9148 genes on average). For comparison, 14,931 genes were detected with FPKM ≥1 in the four rRNA-depleted total RNA samples from bulk HEK293T cells, and 15,535 genes were detected in the four oligo(dT)-enriched total RNA samples from bulk HEK293T cells

    Article Snippet: Sequencing library generation for bulk amount of mouse ES cell RNA and HEK293T cell RNA Total RNA (1 μg) was used for deep-sequencing library construction following the instructions of the TruSeq RNA sample preparation kit (Illumina).

    Techniques: Sequencing, Synthesized, Amplification, Polymerase Chain Reaction, Purification, Sample Prep, RNA Sequencing Assay

    Profiling the transcriptome of V . cholerae in response to polymyxin B exposure using Illumina-based RNA-Seq

    Journal: PLoS ONE

    Article Title: A putative Vibrio cholerae two-component system controls a conserved periplasmic protein in response to the antimicrobial peptide polymyxin B

    doi: 10.1371/journal.pone.0186199

    Figure Lengend Snippet: Profiling the transcriptome of V . cholerae in response to polymyxin B exposure using Illumina-based RNA-Seq

    Article Snippet: Double stranded cDNA ends were repaired and adenylated as described in the Illumina Truseq™ RNA sample preparation low throughput (LT) protocol (Illumina, San Diego, CA).

    Techniques: RNA Sequencing Assay

    Profiling the transcriptome of V . cholerae in response to polymyxin B exposure using Illumina-based RNA-Seq

    Journal: PLoS ONE

    Article Title: A putative Vibrio cholerae two-component system controls a conserved periplasmic protein in response to the antimicrobial peptide polymyxin B

    doi: 10.1371/journal.pone.0186199

    Figure Lengend Snippet: Profiling the transcriptome of V . cholerae in response to polymyxin B exposure using Illumina-based RNA-Seq

    Article Snippet: Double stranded cDNA ends were repaired and adenylated as described in the Illumina Truseq™ RNA sample preparation low throughput (LT) protocol (Illumina, San Diego, CA).

    Techniques: RNA Sequencing Assay

    Summary of the different steps performed in the nextPARS protocol. From the cells or tissue of interest ( A ), total RNA is extracted ( B ) and then poly(A) + RNA is selected ( C ) to initially prepare the samples for nextPARS analyses. Once the quality and quantity of poly(A) + RNA samples is confirmed, RNA samples are denatured and in vitro folded to perform the enzymatic probing of the molecules with the corresponding concentrations of RNase V1 and S1 nuclease ( D ). For the library preparation using the Illumina TruSeq Small RNA Sample Preparation Kit, an initial phosphatase treatment of the 3′ends and a kinase treatment of the 5′ ends are required ( E ) to then ligate the corresponding 5′ and 3′ adapters at the ends of the RNA fragments ( F ). Then a reverse transcription of the RNA fragments and a PCR amplification are performed to obtain the library ( G ). The library is size-selected to get rid of primers and adapters dimers using an acrylamide gel and a final quality control is performed ( H ). Libraries are sequenced in single-reads with read lengths of 50 nucleotides (nt) using Illumina sequencing platforms ( I ) and computational analyses are done as described in the Materials and Methods section in order to map Illumina reads and determine the enzymatic cleavage points, using the first nucleotide in the 5′ end of the reads (which correspond to the 5′end of original RNA fragments) ( J ).

    Journal: RNA

    Article Title: nextPARS: parallel probing of RNA structures in Illumina

    doi: 10.1261/rna.063073.117

    Figure Lengend Snippet: Summary of the different steps performed in the nextPARS protocol. From the cells or tissue of interest ( A ), total RNA is extracted ( B ) and then poly(A) + RNA is selected ( C ) to initially prepare the samples for nextPARS analyses. Once the quality and quantity of poly(A) + RNA samples is confirmed, RNA samples are denatured and in vitro folded to perform the enzymatic probing of the molecules with the corresponding concentrations of RNase V1 and S1 nuclease ( D ). For the library preparation using the Illumina TruSeq Small RNA Sample Preparation Kit, an initial phosphatase treatment of the 3′ends and a kinase treatment of the 5′ ends are required ( E ) to then ligate the corresponding 5′ and 3′ adapters at the ends of the RNA fragments ( F ). Then a reverse transcription of the RNA fragments and a PCR amplification are performed to obtain the library ( G ). The library is size-selected to get rid of primers and adapters dimers using an acrylamide gel and a final quality control is performed ( H ). Libraries are sequenced in single-reads with read lengths of 50 nucleotides (nt) using Illumina sequencing platforms ( I ) and computational analyses are done as described in the Materials and Methods section in order to map Illumina reads and determine the enzymatic cleavage points, using the first nucleotide in the 5′ end of the reads (which correspond to the 5′end of original RNA fragments) ( J ).

    Article Snippet: One of the main limitations in the experimental protocol of nextPARS is that, when using the Illumina TruSeq Small RNA Kit to prepare the libraries, we cannot directly ligate first the 5′ adaptor and then the 3′ adaptor, requiring the performance of phosphatase and kinase treatments of the RNA ends.

    Techniques: In Vitro, Sample Prep, Polymerase Chain Reaction, Amplification, Acrylamide Gel Assay, Sequencing

    RNA-seq profiling of individual kdm2aa -deficient embryos and their siblings. (A) Summary of experimental design. TruSeq stranded mRNA sequencing libraries were prepared from four individual embryos from each genotype at both 5 d.p.f. and 12 d.p.f., totalling 48 libraries. Paired end sequencing with a read length of 75 bp was performed on four lanes of Illumina HiSeq 2500 machines. Sequence was aligned to the GRCz10 reference genome with TopHat and read counts were obtained with HTseq-count. Quality control was performed using QoRTs and outliers removed. 32,261 genes were detected. DESeq2 was used to determine DE genes in each individual clutch, DE genes at each stage by combining the two clutches for each stage whilst controlling for clutch in the DESeq2 model, and also in a combined analysis combining all 4 clutches whilst controlling for stage and clutch. (B) Venn diagram showing the overlap in DE genes between the two 5 d.p.f. experiments and the combined 5 d.p.f. analysis. The increase in power due to the increased sample size enabled an additional 1106 DE genes to be detected. (C) Venn diagram displaying the overlap in DE genes between the two 12 d.p.f. experiments and the combined 12 d.p.f. analysis. An additional 1102 DE genes were detected in the combined analysis. (D) Volcano plot of all detected genes in the combined RNA-seq analysis with the 3752 DE genes shown in red and kdm2aa circled. (E) Box plot of normalised counts for the chromatin modifier nsd1b , with adjusted p-values for individual experiments, stage-specific and combined analysis as indicated by horizontal bars. Data for heterozygous and wild-type siblings are combined. In the figure legend ‘s’ denotes siblings and ‘m’ homozygous mutants. (F) Box plot of normalised counts for dnmt3bb . 2 , a de novo DNA methyl transferase, with adjusted p-values for individual experiments, stage-specific and combined analysis as indicated by horizontal bars. Data for heterozygous and wild-type siblings are combined. In the figure legend ‘s’ denotes siblings and ‘m’ homozygous mutants. (G) Table of enriched Gene Ontology (GO) terms of the biological process (BP) GO domain which overlap in both 5 d.p.f. experiments and also the 5 d.p.f. combined analysis. P-values shown are for the combined analysis. (H) Table of enriched GO terms that overlap in both individual 12 d.p.f. experiments and the 12 d.p.f. combined analysis, with p-values for the combined analysis shown.

    Journal: PLoS Genetics

    Article Title: Loss of the chromatin modifier Kdm2aa causes BrafV600E-independent spontaneous melanoma in zebrafish

    doi: 10.1371/journal.pgen.1006959

    Figure Lengend Snippet: RNA-seq profiling of individual kdm2aa -deficient embryos and their siblings. (A) Summary of experimental design. TruSeq stranded mRNA sequencing libraries were prepared from four individual embryos from each genotype at both 5 d.p.f. and 12 d.p.f., totalling 48 libraries. Paired end sequencing with a read length of 75 bp was performed on four lanes of Illumina HiSeq 2500 machines. Sequence was aligned to the GRCz10 reference genome with TopHat and read counts were obtained with HTseq-count. Quality control was performed using QoRTs and outliers removed. 32,261 genes were detected. DESeq2 was used to determine DE genes in each individual clutch, DE genes at each stage by combining the two clutches for each stage whilst controlling for clutch in the DESeq2 model, and also in a combined analysis combining all 4 clutches whilst controlling for stage and clutch. (B) Venn diagram showing the overlap in DE genes between the two 5 d.p.f. experiments and the combined 5 d.p.f. analysis. The increase in power due to the increased sample size enabled an additional 1106 DE genes to be detected. (C) Venn diagram displaying the overlap in DE genes between the two 12 d.p.f. experiments and the combined 12 d.p.f. analysis. An additional 1102 DE genes were detected in the combined analysis. (D) Volcano plot of all detected genes in the combined RNA-seq analysis with the 3752 DE genes shown in red and kdm2aa circled. (E) Box plot of normalised counts for the chromatin modifier nsd1b , with adjusted p-values for individual experiments, stage-specific and combined analysis as indicated by horizontal bars. Data for heterozygous and wild-type siblings are combined. In the figure legend ‘s’ denotes siblings and ‘m’ homozygous mutants. (F) Box plot of normalised counts for dnmt3bb . 2 , a de novo DNA methyl transferase, with adjusted p-values for individual experiments, stage-specific and combined analysis as indicated by horizontal bars. Data for heterozygous and wild-type siblings are combined. In the figure legend ‘s’ denotes siblings and ‘m’ homozygous mutants. (G) Table of enriched Gene Ontology (GO) terms of the biological process (BP) GO domain which overlap in both 5 d.p.f. experiments and also the 5 d.p.f. combined analysis. P-values shown are for the combined analysis. (H) Table of enriched GO terms that overlap in both individual 12 d.p.f. experiments and the 12 d.p.f. combined analysis, with p-values for the combined analysis shown.

    Article Snippet: From these 48 samples 300 ng total RNA were used to prepare sequencing libraries with Ambion ERCC spike-in mix 1 (Cat. No. 4456740) according to the manufacturer’s instructions using the Illumina TruSeq Stranded mRNA Sample Prep Kit Set A and B (RS-122-2101 and RS-122-2102).

    Techniques: RNA Sequencing Assay, Sequencing

    gas6 may only have subtle roles in caudal hindbrain development. a WT and gas6 mutant embryos were assayed for expression of valentino (r5/r6) (i), hoxb3a (r5-spinal cord) (ii), hoxa3 (r5/r6) (iii), islet1 (cranial nerves)(iv) and dm20 (oligodendrocyte marker) (vi) by ISH, as well as for the presence of OPCs and abducens neurons by crossing to the Tg (olig2:EGFP) vu12 line (v). In column (iv), yellow brackets mark cranial nerve V, blue brackets mark cranial nerve VII and red brackets mark cranial nerve X. White brackets indicate the presence of abducens (cranial nerve VI) in column (v). b Schemes showing RNA-seq library synthesis. Hindbrain tissue was dissected from 48 hpf gas6 mutant embryos in the olig2:eGFP background. Total RNA was collected from pools of hindbrain tissue and was used in library synthesis following the TruSeq Stranded mRNA Library Prep Kit (Illumina) protocol. c 1590 differentially expressed genes were identified from RNA-Seq where 41 out of the 928 up-regulated genes and 78 out of the 662 down-regulated genes were expressed in the hindbrain. GO terms related to Biological Processes were identified in both up-regulated and down-regulated genes using DAVID. d A subset of differentially expressed genes was validated via qPCR from independently collected hindbrain tissue samples. e ISH analysis of representative differentially expressed hindbrain genes (i) neurod6b , (ii) atoh1b and (iii) olig4 show no detectable change in expression pattern in gas6 mutant embryos

    Journal: Neural Development

    Article Title: Analysis of novel caudal hindbrain genes reveals different regulatory logic for gene expression in rhombomere 4 versus 5/6 in embryonic zebrafish

    doi: 10.1186/s13064-018-0112-y

    Figure Lengend Snippet: gas6 may only have subtle roles in caudal hindbrain development. a WT and gas6 mutant embryos were assayed for expression of valentino (r5/r6) (i), hoxb3a (r5-spinal cord) (ii), hoxa3 (r5/r6) (iii), islet1 (cranial nerves)(iv) and dm20 (oligodendrocyte marker) (vi) by ISH, as well as for the presence of OPCs and abducens neurons by crossing to the Tg (olig2:EGFP) vu12 line (v). In column (iv), yellow brackets mark cranial nerve V, blue brackets mark cranial nerve VII and red brackets mark cranial nerve X. White brackets indicate the presence of abducens (cranial nerve VI) in column (v). b Schemes showing RNA-seq library synthesis. Hindbrain tissue was dissected from 48 hpf gas6 mutant embryos in the olig2:eGFP background. Total RNA was collected from pools of hindbrain tissue and was used in library synthesis following the TruSeq Stranded mRNA Library Prep Kit (Illumina) protocol. c 1590 differentially expressed genes were identified from RNA-Seq where 41 out of the 928 up-regulated genes and 78 out of the 662 down-regulated genes were expressed in the hindbrain. GO terms related to Biological Processes were identified in both up-regulated and down-regulated genes using DAVID. d A subset of differentially expressed genes was validated via qPCR from independently collected hindbrain tissue samples. e ISH analysis of representative differentially expressed hindbrain genes (i) neurod6b , (ii) atoh1b and (iii) olig4 show no detectable change in expression pattern in gas6 mutant embryos

    Article Snippet: For each RNA-seq experiment, three libraries were synthesized from 3μg RNA for each WT and mutant sample using the TruSeq Stranded mRNA Library Prep Kit (Illumina).

    Techniques: Mutagenesis, Expressing, Marker, In Situ Hybridization, RNA Sequencing Assay, Real-time Polymerase Chain Reaction

    Comparative schematic of small RNA barcoding methods. The three methods start with ligation of a 3' and 5' RNA adapter to generate a substrate for RT-PCR. In the pre-PCR barcoding method, the barcode is incorporated in the 5' adapter. In the TruSeq method, the barcode is incorporated in one of the RT-PCR primers. In the PALM barcoding method, the amplified RT-PCR product is A-tailed and ligated to a T-tailed barcoded adapter.

    Journal: PLoS ONE

    Article Title: Quantitative Bias in Illumina TruSeq and a Novel Post Amplification Barcoding Strategy for Multiplexed DNA and Small RNA Deep Sequencing

    doi: 10.1371/journal.pone.0026969

    Figure Lengend Snippet: Comparative schematic of small RNA barcoding methods. The three methods start with ligation of a 3' and 5' RNA adapter to generate a substrate for RT-PCR. In the pre-PCR barcoding method, the barcode is incorporated in the 5' adapter. In the TruSeq method, the barcode is incorporated in one of the RT-PCR primers. In the PALM barcoding method, the amplified RT-PCR product is A-tailed and ligated to a T-tailed barcoded adapter.

    Article Snippet: TruSeq small RNA and DNA barcoding and sequencing For the TruSeq sample preparation, the Illumina TruSeq Small RNA Sample Prep Kit (RS-200–0012) and the Illumina TruSeq DNA Sample Prep Kit (FC-121–1001) were used.

    Techniques: Ligation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification

    Adapter-tRNA ligation efficiencies with a conventional method and YAMAT-seq. ( A ) Schematic representation of the adapter ligation reactions used in the conventional and YAMAT-seq methods. In the conventional method, tRNA was subjected to 3΄-adapter (3΄-AD) and 5΄-adapter (5΄-AD) ligations catalyzed by truncated Rnl2 and Rnl1, respectively, using the Illumina TruSeq Small RNA Sample Preparation Kit. In contrast, in the YAMAT-seq method, a Y-shaped adapter (Y-AD) is ligated to a tRNA by Rnl2. ( B ) Ligation products of synthetic human cyto tRNA AspGUC and 5΄-AD or 3΄-AD were quantified by real-time qRT-PCR. The amounts of ligation products were determined based on standard curves, and ligation efficiencies were calculated as percentages of ligated tRNA versus input tRNA (set as 100%). Each data set represents the average of three independent experiments with bars showing the SD. ( C ) Ligation products of the indicated native cyto tRNAs in BT-474 total RNA and 5΄-AD or 3΄-AD were quantified by real-time qRT-PCR. Each data set represents the average Ct values of three independent experiments with bars showing the SD. ( D ) Image of 8% native polyacrylamide gel electrophoresis (PAGE) of amplified cDNAs resulting from BT-474 total RNA sequencing by conventional and YAMAT-seq procedures. The total adapter lengths of the conventional and YAMAT-seq procedures are 118 and 140 bp, respectively. The YAMAT-seq-derived cDNAs in the designated Bands 1–3 regions were gel-purified and subjected to cloning; the identified sequences are shown in Supplementary Table S1 . The cDNAs in regions designated with gray lines are expected to contain tRNA fraction.

    Journal: Nucleic Acids Research

    Article Title: YAMAT-seq: an efficient method for high-throughput sequencing of mature transfer RNAs

    doi: 10.1093/nar/gkx005

    Figure Lengend Snippet: Adapter-tRNA ligation efficiencies with a conventional method and YAMAT-seq. ( A ) Schematic representation of the adapter ligation reactions used in the conventional and YAMAT-seq methods. In the conventional method, tRNA was subjected to 3΄-adapter (3΄-AD) and 5΄-adapter (5΄-AD) ligations catalyzed by truncated Rnl2 and Rnl1, respectively, using the Illumina TruSeq Small RNA Sample Preparation Kit. In contrast, in the YAMAT-seq method, a Y-shaped adapter (Y-AD) is ligated to a tRNA by Rnl2. ( B ) Ligation products of synthetic human cyto tRNA AspGUC and 5΄-AD or 3΄-AD were quantified by real-time qRT-PCR. The amounts of ligation products were determined based on standard curves, and ligation efficiencies were calculated as percentages of ligated tRNA versus input tRNA (set as 100%). Each data set represents the average of three independent experiments with bars showing the SD. ( C ) Ligation products of the indicated native cyto tRNAs in BT-474 total RNA and 5΄-AD or 3΄-AD were quantified by real-time qRT-PCR. Each data set represents the average Ct values of three independent experiments with bars showing the SD. ( D ) Image of 8% native polyacrylamide gel electrophoresis (PAGE) of amplified cDNAs resulting from BT-474 total RNA sequencing by conventional and YAMAT-seq procedures. The total adapter lengths of the conventional and YAMAT-seq procedures are 118 and 140 bp, respectively. The YAMAT-seq-derived cDNAs in the designated Bands 1–3 regions were gel-purified and subjected to cloning; the identified sequences are shown in Supplementary Table S1 . The cDNAs in regions designated with gray lines are expected to contain tRNA fraction.

    Article Snippet: cDNA amplification and Illumina sequencing cDNA amplification was performed using the TruSeq Small RNA Sample Preparation Kit (Illumina) according to the manufacturer's protocol.

    Techniques: Ligation, Sample Prep, Quantitative RT-PCR, Polyacrylamide Gel Electrophoresis, Amplification, RNA Sequencing Assay, Derivative Assay, Purification, Clone Assay

    Polyomavirus genome and work flow. (A) Polyomavirus genome including origin and polyadenylation sites. Early gene splicing shown in blue. Late gene splicing shown in red. (B) Schematic of read-through of the polyadenylation site during the late phase of infection. Late transcripts must read through the entire viral genome at least once to allow for the late leader exon (L) to splice properly. This results in spliced late mRNAs with at least two tandem repeats of the late leader exon. (C) Work flow of experiments. NIH 3T6 cells were infected with the Py59RA strain of polyomavirus and either harvested at different time points or treated with aphidicolin to block DNA replication and keep the infection in the early phase for 48 hours. Total RNA was collected and used to synthesize stranded cDNA libraries using the Illumina TruSeq Stranded Total RNA Preparation kit. Samples were run on the Hiseq 2000 sequencer and aligned to both the Py59RA and mouse host genomes.

    Journal: PLoS Pathogens

    Article Title: Global Analysis of Mouse Polyomavirus Infection Reveals Dynamic Regulation of Viral and Host Gene Expression and Promiscuous Viral RNA Editing

    doi: 10.1371/journal.ppat.1005166

    Figure Lengend Snippet: Polyomavirus genome and work flow. (A) Polyomavirus genome including origin and polyadenylation sites. Early gene splicing shown in blue. Late gene splicing shown in red. (B) Schematic of read-through of the polyadenylation site during the late phase of infection. Late transcripts must read through the entire viral genome at least once to allow for the late leader exon (L) to splice properly. This results in spliced late mRNAs with at least two tandem repeats of the late leader exon. (C) Work flow of experiments. NIH 3T6 cells were infected with the Py59RA strain of polyomavirus and either harvested at different time points or treated with aphidicolin to block DNA replication and keep the infection in the early phase for 48 hours. Total RNA was collected and used to synthesize stranded cDNA libraries using the Illumina TruSeq Stranded Total RNA Preparation kit. Samples were run on the Hiseq 2000 sequencer and aligned to both the Py59RA and mouse host genomes.

    Article Snippet: Total RNA was harvested at 12, 18, 24, and 36-hours post infection from three biological replicates for each condition and was used in the synthesis of stranded cDNA sequencing libraries using the Illumina TruSeq Stranded Total RNA Sample Preparation Kit.

    Techniques: Flow Cytometry, Infection, Blocking Assay