genome analyzer iix Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94
    Illumina Inc genome analyzer iix
    Ago HITS-CLIP analysis of stromal and endothelial cells. (A): Basic experimental scheme of Ago HITS-CLIP as adapted from Chi et al. UV cross-linked cells are lyzed and treated with RNase to reduce the length of mRNA fragments. Ago is immune-precipitated with monoclonal anti-Ago antibody 2A8, P32-labeled 3′ RNA linker ligated (on-bead), and resolved by SDS-PAGE electrophoresis. The protein-RNA-complex is transferred to nitrocellulose and visualized by autoradiography. In the representative autoradiograph shown, a major band is seen ~110 kD and a more diffuse band at ~130 kD. The RNA-protein complex in the 110–130 kD region is isolated and a 5′ RNA linker is ligated. The isolated RNA is reverse transcribed and PCR amplified with <t>Illumina-compatible</t> primers to generate a cDNA library (typical agarose gel shown). The cDNA libraries are then gel purified and sequenced on Illumina GA <t>IIx</t> and resultant bioinformatically analyzed. (B): Distribution of aligned reads from HS27A samples to different gene annotations. (C): Correlation of consensus peaks between HS5 and HS27A. Coefficient of determination ( R 2 determined by corrplot [R-package]). (D): Correlation of consensus peaks between HS5 and hMSC. (E): Correlation of consensus peaks between HS27A and hMSC. (F): Correlation of consensus peaks between TrHBMEC and HUVEC. (G): Overlap of genes enriched by consensus peaks between stromal cells (HS5, HS27A, and hMSC). (H): Overlap of genes enriched by consensus peaks between endothelial cells (TrHBMEC and HUVEC). Abbreviations: hMSC, human mesenchymal stromal cell; HUVEC, human umbilical vein endothelial cell; TrHBMEC, transformed human bone marrow endothelial cells; UV, ultraviolet.
    Genome Analyzer Iix, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 14014 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/genome analyzer iix/product/Illumina Inc
    Average 94 stars, based on 14014 article reviews
    Price from $9.99 to $1999.99
    genome analyzer iix - by Bioz Stars, 2020-11
    94/100 stars
      Buy from Supplier

    94
    Illumina Inc illumina gaiix platform
    Comparison of miRNA expression profiles among different library preparation protocols reveals massive differential bias. A comparison of the following four methods is shown: Illumina v1.5 library preparation sequenced on <t>GAIIx</t> platform (v1.5_GAIIx), Illumina TruSeq library preparation sequenced on GAIIx platform (TS_GAIIx), Illumina TruSeq library preparation sequenced on HiSeq platform (TS_HiSeq) and Bioo Scientific NEXTflexV2 library preparation sequenced on the HiSeq platform (NF-HiSeq). Three biological replicate small <t>RNA</t> libraries were generated for each of the first three methods and one replicate was generated for the NF-HiSeq method. (A) Correlation of miRNA profiles between each pair of datasets (correlation values were calculated by Pearson’s metric). Similar results were obtained with Spearman’s correlation coefficient, rho (data not shown). White and blue colors indicate strongest and weakest correlation, respectively. (B) miRNA expression profiles across all 10 samples. Hierarchical clustering was used to identify samples with closely related expression profiles. Expression is represented as z -score, indicating the number of standard deviations below (purple) or above (orange) the mean across all ten libraries. Both (A,B) used only the set of miRNAs identified as “highly expressed” ( n = 358).
    Illumina Gaiix Platform, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 3290 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/illumina gaiix platform/product/Illumina Inc
    Average 94 stars, based on 3290 article reviews
    Price from $9.99 to $1999.99
    illumina gaiix platform - by Bioz Stars, 2020-11
    94/100 stars
      Buy from Supplier

    94
    Illumina Inc illumina gaiix sequencer
    RNA-protein interactions can be assayed by HiTS-RAP on an <t>Illumina</t> <t>GAIIx</t> instrument ( a ) HiTS-RAP schematic. Sequencing is done following the standard Illumina workflow. The strand synthesized during sequencing is then stripped away, a primer is annealed and the second strand is regenerated with Klenow enzyme. Tus is then bound to the ter site, and DNAs on the flowcell are transcribed. T7 RNAP initiates at its promoter, transcribes through the sequence of interest, and halts just upstream of the Tus bound ter site. The RNA transcript is stably linked to its DNA template through the polymerase. Fluorescently labeled protein is then bound to the RNA and imaged. ( b ) Images from a HiTS-RAP run with GFP and SRB-2 aptamers. ‘All clusters’ (left panels) are labeled during sequencing and shown as a maximum intensity projection of the four channels. After transcription halting and EGFP-mOrange binding, the flowcell is imaged at 625 nM EGFP-mOrange. GFPapt clusters are labeled by mOrange while SRB-2 aptamer clusters are not. Scale bars: 6.75 μM, 1.125 μM in inset ( c ) Binding curves for the GFP aptamer ( n = 2,665,064), and mutants C58U ( n = 3,833), C76U ( n = 4,758), and G8U_U56C ( n = 29), and the SRB-2 aptamer ( n = 1,588,404). G8U_U56C and SRB-2 aptamer are scored as not binding. Data are from one lane of the sequencer (SRB-2 aptamer is from a separate lane). Intensities are the average of all clusters of each sequence in the lane, normalized by dividing by their average sequencing intensity and subtracting their average intensity at no EGFP-mOrange. Error bars represent standard error. Error of fitted K d s are the square root of variances returned by the fitting algorithm. ( d ) EMSA of RNAs in part c. K d s are determined from a single fit to two replicate EMSA experiments for each sequence, ± standard deviation fitted by IGOR. The G8U_U56C gel was also scanned to visualize EGFP.
    Illumina Gaiix Sequencer, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 1680 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/illumina gaiix sequencer/product/Illumina Inc
    Average 94 stars, based on 1680 article reviews
    Price from $9.99 to $1999.99
    illumina gaiix sequencer - by Bioz Stars, 2020-11
    94/100 stars
      Buy from Supplier

    90
    Illumina Inc genome analyzer gaiix
    Schematic diagram of the workflow for O. melastigma miRNA discovery. Brain, liver and gonadal (ovary and testis) tissues of male and female marine medaka were submitted to small RNA libraries preparation and were sequenced using <t>Illumina</t> <t>GAIIX.</t> After the removal of low quality reads and filtering, high quality sequencing reads were blasted against miRBase v.17 to identify the conserved miRNAs of marine medaka.
    Genome Analyzer Gaiix, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 90/100, based on 694 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/genome analyzer gaiix/product/Illumina Inc
    Average 90 stars, based on 694 article reviews
    Price from $9.99 to $1999.99
    genome analyzer gaiix - by Bioz Stars, 2020-11
    90/100 stars
      Buy from Supplier

    91
    Illumina Inc illumina gaiix machine
    Schematic diagram of the workflow for O. melastigma miRNA discovery. Brain, liver and gonadal (ovary and testis) tissues of male and female marine medaka were submitted to small RNA libraries preparation and were sequenced using <t>Illumina</t> <t>GAIIX.</t> After the removal of low quality reads and filtering, high quality sequencing reads were blasted against miRBase v.17 to identify the conserved miRNAs of marine medaka.
    Illumina Gaiix Machine, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 91/100, based on 245 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/illumina gaiix machine/product/Illumina Inc
    Average 91 stars, based on 245 article reviews
    Price from $9.99 to $1999.99
    illumina gaiix machine - by Bioz Stars, 2020-11
    91/100 stars
      Buy from Supplier

    88
    Solexa solexa gaiix
    Schematic diagram of the workflow for O. melastigma miRNA discovery. Brain, liver and gonadal (ovary and testis) tissues of male and female marine medaka were submitted to small RNA libraries preparation and were sequenced using <t>Illumina</t> <t>GAIIX.</t> After the removal of low quality reads and filtering, high quality sequencing reads were blasted against miRBase v.17 to identify the conserved miRNAs of marine medaka.
    Solexa Gaiix, supplied by Solexa, used in various techniques. Bioz Stars score: 88/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/solexa gaiix/product/Solexa
    Average 88 stars, based on 34 article reviews
    Price from $9.99 to $1999.99
    solexa gaiix - by Bioz Stars, 2020-11
    88/100 stars
      Buy from Supplier

    89
    Illumina Inc gaiix flow cell
    Schematic diagram of the workflow for O. melastigma miRNA discovery. Brain, liver and gonadal (ovary and testis) tissues of male and female marine medaka were submitted to small RNA libraries preparation and were sequenced using <t>Illumina</t> <t>GAIIX.</t> After the removal of low quality reads and filtering, high quality sequencing reads were blasted against miRBase v.17 to identify the conserved miRNAs of marine medaka.
    Gaiix Flow Cell, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 89/100, based on 91 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gaiix flow cell/product/Illumina Inc
    Average 89 stars, based on 91 article reviews
    Price from $9.99 to $1999.99
    gaiix flow cell - by Bioz Stars, 2020-11
    89/100 stars
      Buy from Supplier

    88
    Illumina Inc gaiix dna sequencer
    Schematic diagram of the workflow for O. melastigma miRNA discovery. Brain, liver and gonadal (ovary and testis) tissues of male and female marine medaka were submitted to small RNA libraries preparation and were sequenced using <t>Illumina</t> <t>GAIIX.</t> After the removal of low quality reads and filtering, high quality sequencing reads were blasted against miRBase v.17 to identify the conserved miRNAs of marine medaka.
    Gaiix Dna Sequencer, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 88/100, based on 62 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gaiix dna sequencer/product/Illumina Inc
    Average 88 stars, based on 62 article reviews
    Price from $9.99 to $1999.99
    gaiix dna sequencer - by Bioz Stars, 2020-11
    88/100 stars
      Buy from Supplier

    Image Search Results


    Ago HITS-CLIP analysis of stromal and endothelial cells. (A): Basic experimental scheme of Ago HITS-CLIP as adapted from Chi et al. UV cross-linked cells are lyzed and treated with RNase to reduce the length of mRNA fragments. Ago is immune-precipitated with monoclonal anti-Ago antibody 2A8, P32-labeled 3′ RNA linker ligated (on-bead), and resolved by SDS-PAGE electrophoresis. The protein-RNA-complex is transferred to nitrocellulose and visualized by autoradiography. In the representative autoradiograph shown, a major band is seen ~110 kD and a more diffuse band at ~130 kD. The RNA-protein complex in the 110–130 kD region is isolated and a 5′ RNA linker is ligated. The isolated RNA is reverse transcribed and PCR amplified with Illumina-compatible primers to generate a cDNA library (typical agarose gel shown). The cDNA libraries are then gel purified and sequenced on Illumina GA IIx and resultant bioinformatically analyzed. (B): Distribution of aligned reads from HS27A samples to different gene annotations. (C): Correlation of consensus peaks between HS5 and HS27A. Coefficient of determination ( R 2 determined by corrplot [R-package]). (D): Correlation of consensus peaks between HS5 and hMSC. (E): Correlation of consensus peaks between HS27A and hMSC. (F): Correlation of consensus peaks between TrHBMEC and HUVEC. (G): Overlap of genes enriched by consensus peaks between stromal cells (HS5, HS27A, and hMSC). (H): Overlap of genes enriched by consensus peaks between endothelial cells (TrHBMEC and HUVEC). Abbreviations: hMSC, human mesenchymal stromal cell; HUVEC, human umbilical vein endothelial cell; TrHBMEC, transformed human bone marrow endothelial cells; UV, ultraviolet.

    Journal: Stem cells (Dayton, Ohio)

    Article Title: Genome-Wide Analysis of miRNA-mRNA Interactions in Marrow Stromal Cells

    doi: 10.1002/stem.1531

    Figure Lengend Snippet: Ago HITS-CLIP analysis of stromal and endothelial cells. (A): Basic experimental scheme of Ago HITS-CLIP as adapted from Chi et al. UV cross-linked cells are lyzed and treated with RNase to reduce the length of mRNA fragments. Ago is immune-precipitated with monoclonal anti-Ago antibody 2A8, P32-labeled 3′ RNA linker ligated (on-bead), and resolved by SDS-PAGE electrophoresis. The protein-RNA-complex is transferred to nitrocellulose and visualized by autoradiography. In the representative autoradiograph shown, a major band is seen ~110 kD and a more diffuse band at ~130 kD. The RNA-protein complex in the 110–130 kD region is isolated and a 5′ RNA linker is ligated. The isolated RNA is reverse transcribed and PCR amplified with Illumina-compatible primers to generate a cDNA library (typical agarose gel shown). The cDNA libraries are then gel purified and sequenced on Illumina GA IIx and resultant bioinformatically analyzed. (B): Distribution of aligned reads from HS27A samples to different gene annotations. (C): Correlation of consensus peaks between HS5 and HS27A. Coefficient of determination ( R 2 determined by corrplot [R-package]). (D): Correlation of consensus peaks between HS5 and hMSC. (E): Correlation of consensus peaks between HS27A and hMSC. (F): Correlation of consensus peaks between TrHBMEC and HUVEC. (G): Overlap of genes enriched by consensus peaks between stromal cells (HS5, HS27A, and hMSC). (H): Overlap of genes enriched by consensus peaks between endothelial cells (TrHBMEC and HUVEC). Abbreviations: hMSC, human mesenchymal stromal cell; HUVEC, human umbilical vein endothelial cell; TrHBMEC, transformed human bone marrow endothelial cells; UV, ultraviolet.

    Article Snippet: Sequencing was performed on an Illumina Genome Analyzer IIx (single end 50 base pair reads).

    Techniques: Cross-linking Immunoprecipitation, Labeling, SDS Page, Electrophoresis, Autoradiography, Isolation, Polymerase Chain Reaction, Amplification, cDNA Library Assay, Agarose Gel Electrophoresis, Purification, Transformation Assay

    Comparison of miRNA expression profiles among different library preparation protocols reveals massive differential bias. A comparison of the following four methods is shown: Illumina v1.5 library preparation sequenced on GAIIx platform (v1.5_GAIIx), Illumina TruSeq library preparation sequenced on GAIIx platform (TS_GAIIx), Illumina TruSeq library preparation sequenced on HiSeq platform (TS_HiSeq) and Bioo Scientific NEXTflexV2 library preparation sequenced on the HiSeq platform (NF-HiSeq). Three biological replicate small RNA libraries were generated for each of the first three methods and one replicate was generated for the NF-HiSeq method. (A) Correlation of miRNA profiles between each pair of datasets (correlation values were calculated by Pearson’s metric). Similar results were obtained with Spearman’s correlation coefficient, rho (data not shown). White and blue colors indicate strongest and weakest correlation, respectively. (B) miRNA expression profiles across all 10 samples. Hierarchical clustering was used to identify samples with closely related expression profiles. Expression is represented as z -score, indicating the number of standard deviations below (purple) or above (orange) the mean across all ten libraries. Both (A,B) used only the set of miRNAs identified as “highly expressed” ( n = 358).

    Journal: Frontiers in Genetics

    Article Title: Addressing Bias in Small RNA Library Preparation for Sequencing: A New Protocol Recovers MicroRNAs that Evade Capture by Current Methods

    doi: 10.3389/fgene.2015.00352

    Figure Lengend Snippet: Comparison of miRNA expression profiles among different library preparation protocols reveals massive differential bias. A comparison of the following four methods is shown: Illumina v1.5 library preparation sequenced on GAIIx platform (v1.5_GAIIx), Illumina TruSeq library preparation sequenced on GAIIx platform (TS_GAIIx), Illumina TruSeq library preparation sequenced on HiSeq platform (TS_HiSeq) and Bioo Scientific NEXTflexV2 library preparation sequenced on the HiSeq platform (NF-HiSeq). Three biological replicate small RNA libraries were generated for each of the first three methods and one replicate was generated for the NF-HiSeq method. (A) Correlation of miRNA profiles between each pair of datasets (correlation values were calculated by Pearson’s metric). Similar results were obtained with Spearman’s correlation coefficient, rho (data not shown). White and blue colors indicate strongest and weakest correlation, respectively. (B) miRNA expression profiles across all 10 samples. Hierarchical clustering was used to identify samples with closely related expression profiles. Expression is represented as z -score, indicating the number of standard deviations below (purple) or above (orange) the mean across all ten libraries. Both (A,B) used only the set of miRNAs identified as “highly expressed” ( n = 358).

    Article Snippet: Results We isolated RNA from a widely used pancreatic beta-cell-like cell line (MIN6) and performed small RNA-seq using four different methods: (1) Illumina v1.5 library preparation sequenced on GAIIx platform (v1.5-GAIIx), (2) Illumina TruSeq library preparation sequenced on GAIIx platform (TS-GAIIx), (3) Illumina TruSeq library preparation sequenced on HiSeq platform (TS-HiSeq), and (4) Bioo Scientific NEXTflex V2 library preparation sequenced on the HiSeq platform (NF-HiSeq).

    Techniques: Expressing, Generated

    Fifty of the most abundant miRNAs are greater than ten-fold differentially detected between Illumina v1.5 and TruSeq. (A) Comparison of relative expression levels of miRNAs in MIN6 ( n = 358) between the GAIIx and HiSeq sequencing platforms with libraries prepared by TruSeq (TS) is shown. Each data point represents the average relative expression level for an individual miRNA across three biological replicates. (B) Comparison of relative expression levels of miRNAs in MIN6 ( n = 358) between the v1.5 and TruSeq (TS) library preparation methods is shown. Each data point represents the average relative expression level for an individual miRNA across three biological replicates. (C,D) Comparison of relative expression levels of miRNAs in MIN6 ( n = 358, C ) and mouse liver ( n = 178, D ) between the TruSeq (TS) and NEXTflex (NF) library preparation methods is shown. Each data point represents the average relative expression level for an individual miRNA across three biological replicates (A,B) , or one biological replicate (C,D) . Relative miRNA expression levels were calculated according to the following: log 10 (mean(miRNA RPMM)), where RPMM is reads per million mapped reads. Pearson correlation values are displayed in red text within each panel, and gray dashed lines denote 10-fold differential expression.

    Journal: Frontiers in Genetics

    Article Title: Addressing Bias in Small RNA Library Preparation for Sequencing: A New Protocol Recovers MicroRNAs that Evade Capture by Current Methods

    doi: 10.3389/fgene.2015.00352

    Figure Lengend Snippet: Fifty of the most abundant miRNAs are greater than ten-fold differentially detected between Illumina v1.5 and TruSeq. (A) Comparison of relative expression levels of miRNAs in MIN6 ( n = 358) between the GAIIx and HiSeq sequencing platforms with libraries prepared by TruSeq (TS) is shown. Each data point represents the average relative expression level for an individual miRNA across three biological replicates. (B) Comparison of relative expression levels of miRNAs in MIN6 ( n = 358) between the v1.5 and TruSeq (TS) library preparation methods is shown. Each data point represents the average relative expression level for an individual miRNA across three biological replicates. (C,D) Comparison of relative expression levels of miRNAs in MIN6 ( n = 358, C ) and mouse liver ( n = 178, D ) between the TruSeq (TS) and NEXTflex (NF) library preparation methods is shown. Each data point represents the average relative expression level for an individual miRNA across three biological replicates (A,B) , or one biological replicate (C,D) . Relative miRNA expression levels were calculated according to the following: log 10 (mean(miRNA RPMM)), where RPMM is reads per million mapped reads. Pearson correlation values are displayed in red text within each panel, and gray dashed lines denote 10-fold differential expression.

    Article Snippet: Results We isolated RNA from a widely used pancreatic beta-cell-like cell line (MIN6) and performed small RNA-seq using four different methods: (1) Illumina v1.5 library preparation sequenced on GAIIx platform (v1.5-GAIIx), (2) Illumina TruSeq library preparation sequenced on GAIIx platform (TS-GAIIx), (3) Illumina TruSeq library preparation sequenced on HiSeq platform (TS-HiSeq), and (4) Bioo Scientific NEXTflex V2 library preparation sequenced on the HiSeq platform (NF-HiSeq).

    Techniques: Expressing, Sequencing

    SNP discovery pipeline using Illumina GAIIx sequence reads of eight flax genotypes aligned against the whole genome shotgun sequence assembly of CDC Bethune.

    Journal: BMC Genomics

    Article Title: Genome wide SNP discovery in flax through next generation sequencing of reduced representation libraries

    doi: 10.1186/1471-2164-13-684

    Figure Lengend Snippet: SNP discovery pipeline using Illumina GAIIx sequence reads of eight flax genotypes aligned against the whole genome shotgun sequence assembly of CDC Bethune.

    Article Snippet: Illumina sequencing RRL construction from the 350-425bp fraction and Illumina/Solexa sequencing [ ] was performed using Illumina GAIIx sequencing platform (Illumina Inc., San Diego, USA) by the Michael Smith Genome Sciences Centre of the BC Cancer Agency, Genome British Columbia (Vancouver, BC, Canada).

    Techniques: Sequencing

    Relationship of SNP discovery with sequence coverage and read depth in seven flax genotypes. ( A ) Read depth frequency distribution of 55,465 SNP locations identified by alignment of Illumina GAIIx reads of seven genotypes against the CDC Bethune whole genome shotgun sequence assembly. A minimum of three reads per genotype was required for SNP calling. A log scale was used for the number of SNPs because of the disproportion in the 3-50 reads bin. ( B ) Correlation of SNP discovery with sequence coverage expressed as genome equivalents (BT-CDC Bethune, MB-Macbeth, SP-SP2047, UG-UGG5-5, DL-Double Low, CT-Crepitam Tabor, G11-G-1186/94, AT-Atlas).

    Journal: BMC Genomics

    Article Title: Genome wide SNP discovery in flax through next generation sequencing of reduced representation libraries

    doi: 10.1186/1471-2164-13-684

    Figure Lengend Snippet: Relationship of SNP discovery with sequence coverage and read depth in seven flax genotypes. ( A ) Read depth frequency distribution of 55,465 SNP locations identified by alignment of Illumina GAIIx reads of seven genotypes against the CDC Bethune whole genome shotgun sequence assembly. A minimum of three reads per genotype was required for SNP calling. A log scale was used for the number of SNPs because of the disproportion in the 3-50 reads bin. ( B ) Correlation of SNP discovery with sequence coverage expressed as genome equivalents (BT-CDC Bethune, MB-Macbeth, SP-SP2047, UG-UGG5-5, DL-Double Low, CT-Crepitam Tabor, G11-G-1186/94, AT-Atlas).

    Article Snippet: Illumina sequencing RRL construction from the 350-425bp fraction and Illumina/Solexa sequencing [ ] was performed using Illumina GAIIx sequencing platform (Illumina Inc., San Diego, USA) by the Michael Smith Genome Sciences Centre of the BC Cancer Agency, Genome British Columbia (Vancouver, BC, Canada).

    Techniques: Sequencing

    RNA-protein interactions can be assayed by HiTS-RAP on an Illumina GAIIx instrument ( a ) HiTS-RAP schematic. Sequencing is done following the standard Illumina workflow. The strand synthesized during sequencing is then stripped away, a primer is annealed and the second strand is regenerated with Klenow enzyme. Tus is then bound to the ter site, and DNAs on the flowcell are transcribed. T7 RNAP initiates at its promoter, transcribes through the sequence of interest, and halts just upstream of the Tus bound ter site. The RNA transcript is stably linked to its DNA template through the polymerase. Fluorescently labeled protein is then bound to the RNA and imaged. ( b ) Images from a HiTS-RAP run with GFP and SRB-2 aptamers. ‘All clusters’ (left panels) are labeled during sequencing and shown as a maximum intensity projection of the four channels. After transcription halting and EGFP-mOrange binding, the flowcell is imaged at 625 nM EGFP-mOrange. GFPapt clusters are labeled by mOrange while SRB-2 aptamer clusters are not. Scale bars: 6.75 μM, 1.125 μM in inset ( c ) Binding curves for the GFP aptamer ( n = 2,665,064), and mutants C58U ( n = 3,833), C76U ( n = 4,758), and G8U_U56C ( n = 29), and the SRB-2 aptamer ( n = 1,588,404). G8U_U56C and SRB-2 aptamer are scored as not binding. Data are from one lane of the sequencer (SRB-2 aptamer is from a separate lane). Intensities are the average of all clusters of each sequence in the lane, normalized by dividing by their average sequencing intensity and subtracting their average intensity at no EGFP-mOrange. Error bars represent standard error. Error of fitted K d s are the square root of variances returned by the fitting algorithm. ( d ) EMSA of RNAs in part c. K d s are determined from a single fit to two replicate EMSA experiments for each sequence, ± standard deviation fitted by IGOR. The G8U_U56C gel was also scanned to visualize EGFP.

    Journal: Nature methods

    Article Title: Comprehensive Analysis of RNA-Protein Interactions by High Throughput Sequencing-RNA Affinity Profiling

    doi: 10.1038/nmeth.2970

    Figure Lengend Snippet: RNA-protein interactions can be assayed by HiTS-RAP on an Illumina GAIIx instrument ( a ) HiTS-RAP schematic. Sequencing is done following the standard Illumina workflow. The strand synthesized during sequencing is then stripped away, a primer is annealed and the second strand is regenerated with Klenow enzyme. Tus is then bound to the ter site, and DNAs on the flowcell are transcribed. T7 RNAP initiates at its promoter, transcribes through the sequence of interest, and halts just upstream of the Tus bound ter site. The RNA transcript is stably linked to its DNA template through the polymerase. Fluorescently labeled protein is then bound to the RNA and imaged. ( b ) Images from a HiTS-RAP run with GFP and SRB-2 aptamers. ‘All clusters’ (left panels) are labeled during sequencing and shown as a maximum intensity projection of the four channels. After transcription halting and EGFP-mOrange binding, the flowcell is imaged at 625 nM EGFP-mOrange. GFPapt clusters are labeled by mOrange while SRB-2 aptamer clusters are not. Scale bars: 6.75 μM, 1.125 μM in inset ( c ) Binding curves for the GFP aptamer ( n = 2,665,064), and mutants C58U ( n = 3,833), C76U ( n = 4,758), and G8U_U56C ( n = 29), and the SRB-2 aptamer ( n = 1,588,404). G8U_U56C and SRB-2 aptamer are scored as not binding. Data are from one lane of the sequencer (SRB-2 aptamer is from a separate lane). Intensities are the average of all clusters of each sequence in the lane, normalized by dividing by their average sequencing intensity and subtracting their average intensity at no EGFP-mOrange. Error bars represent standard error. Error of fitted K d s are the square root of variances returned by the fitting algorithm. ( d ) EMSA of RNAs in part c. K d s are determined from a single fit to two replicate EMSA experiments for each sequence, ± standard deviation fitted by IGOR. The G8U_U56C gel was also scanned to visualize EGFP.

    Article Snippet: We adapted an Illumina GAIIx sequencer to make several million such measurements with a H igh- T hroughput S equencing – R NA A ffinity P rofiling (HiTS-RAP) assay.

    Techniques: Sequencing, Synthesized, Stable Transfection, Labeling, Sulforhodamine B Assay, Binding Assay, Standard Deviation

    Schematic diagram of the workflow for O. melastigma miRNA discovery. Brain, liver and gonadal (ovary and testis) tissues of male and female marine medaka were submitted to small RNA libraries preparation and were sequenced using Illumina GAIIX. After the removal of low quality reads and filtering, high quality sequencing reads were blasted against miRBase v.17 to identify the conserved miRNAs of marine medaka.

    Journal: PLoS ONE

    Article Title: Identification and Expression Profiling of MicroRNAs in the Brain, Liver and Gonads of Marine Medaka (Oryzias melastigma) and in Response to Hypoxia

    doi: 10.1371/journal.pone.0110698

    Figure Lengend Snippet: Schematic diagram of the workflow for O. melastigma miRNA discovery. Brain, liver and gonadal (ovary and testis) tissues of male and female marine medaka were submitted to small RNA libraries preparation and were sequenced using Illumina GAIIX. After the removal of low quality reads and filtering, high quality sequencing reads were blasted against miRBase v.17 to identify the conserved miRNAs of marine medaka.

    Article Snippet: Sequencing was performed on the Illumina Genome Analyzer GAIIx.

    Techniques: Sequencing

    A sex-specific timecourse of early-embryonic gene expression. (A) Transcription events during early embryogenesis. During the first 8–9 mitotic cycles, almost all RNAs in the embryo are of maternal origin. Zygotic transcription begins at a low level at approximately cycle 10 and becomes widespread by the middle of cycle 14. MSL-mediated dosage compensation begins late in or following cycle 14. (B) Embryos used for mRNA-Seq. Individual embryos in the interphases of cycles 10 to 14 were selected by direct observation of mitosis in embryos containing histone H2Av-RFP and computing nuclear density. Embryos at substages of cycle 14 were selected by observing the extent of progression through cellularization (from proportion of membrane invagination) under light microscopy. Each embryo pictured here was placed into TRIzol reagent immediately after these images were taken, DNA and RNA were extracted, and each sample was genotyped to determine the sex of the embryo. (C) Approximately 100 ng total RNA was obtained from each embryo, and poly-A RNA was processed with an amplification-free protocol optimized for small samples and sequenced on an Illumina GAIIx Genome Analyzer. Data (normalized reads per kb, RPKM) from independently processed individuals of the same stage and same sex, and same stage but different sex were extremely similar, while individuals from different stages showed larger numbers of differences.

    Journal: PLoS Biology

    Article Title: Noncanonical Compensation of Zygotic X Transcription in Early Drosophila melanogaster Development Revealed through Single-Embryo RNA-SeqDoubling the Dose

    doi: 10.1371/journal.pbio.1000590

    Figure Lengend Snippet: A sex-specific timecourse of early-embryonic gene expression. (A) Transcription events during early embryogenesis. During the first 8–9 mitotic cycles, almost all RNAs in the embryo are of maternal origin. Zygotic transcription begins at a low level at approximately cycle 10 and becomes widespread by the middle of cycle 14. MSL-mediated dosage compensation begins late in or following cycle 14. (B) Embryos used for mRNA-Seq. Individual embryos in the interphases of cycles 10 to 14 were selected by direct observation of mitosis in embryos containing histone H2Av-RFP and computing nuclear density. Embryos at substages of cycle 14 were selected by observing the extent of progression through cellularization (from proportion of membrane invagination) under light microscopy. Each embryo pictured here was placed into TRIzol reagent immediately after these images were taken, DNA and RNA were extracted, and each sample was genotyped to determine the sex of the embryo. (C) Approximately 100 ng total RNA was obtained from each embryo, and poly-A RNA was processed with an amplification-free protocol optimized for small samples and sequenced on an Illumina GAIIx Genome Analyzer. Data (normalized reads per kb, RPKM) from independently processed individuals of the same stage and same sex, and same stage but different sex were extremely similar, while individuals from different stages showed larger numbers of differences.

    Article Snippet: We sequenced a total of 24 mRNA samples on an Illumina GAIIx Genome Analyzer.

    Techniques: Expressing, Light Microscopy, Amplification