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Illumina Inc polyadenylated mrna molecules
Polyadenylated Mrna Molecules, 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
https://www.bioz.com/result/polyadenylated mrna molecules/product/Illumina Inc
Average 85 stars, based on 1 article reviews
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polyadenylated mrna molecules - by Bioz Stars, 2020-09
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Amplification:

Article Title: Transcriptome and methylome interactions in rice hybrids
Article Snippet: .. Total RNA (10 ug) for each sample was used for purification of the poly(A)-containing mRNA molecules, RNA amplification, and synthesis of double-stranded cDNAs that were ligated to adapters, thus preparing the libraries for sequencing on the Illumina GAII. .. The RNA-seq library preparation protocol was followed as described in Illumina’s standard protocol for RNA-seq libraries (Illumina).

RNA Sequencing Assay:

Article Title: Comprehensive epigenome characterization reveals diverse transcriptional regulation across human vascular endothelial cells
Article Snippet: .. RNA-seq analysis Poly(A)-containing mRNA molecules were isolated from total RNA and then converted to cDNA with oligo(dT) primers using a TruSeq RNA Sample Preparation kit v2 (Illumina) and were sequenced with a HiSeq 2500 system (Illumina). .. We applied sequenced paired-end reads to kallisto version 0.43.1 [ ] with the “–rf-stranded -b 100” option, which estimates the transcript-level expression values as Transcripts Per Kilobase Million (TPM, Ensembl gene annotation GRCh37).

Magnetic Beads:

Article Title: Genome-Wide Identification of Small RNAs in Bifidobacterium animalis subsp. lactis KLDS 2.0603 and Their Regulation Role in the Adaption to Gastrointestinal Environment
Article Snippet: .. Briefly, the poly-A containing mRNA molecules were purified using poly-T oligo-attached magnetic beads (Illumina, San Diego, CA) from total RNA, and sheared to small fragments using divalent cations under elevated temperature. ..

Isolation:

Article Title: Comprehensive epigenome characterization reveals diverse transcriptional regulation across human vascular endothelial cells
Article Snippet: .. RNA-seq analysis Poly(A)-containing mRNA molecules were isolated from total RNA and then converted to cDNA with oligo(dT) primers using a TruSeq RNA Sample Preparation kit v2 (Illumina) and were sequenced with a HiSeq 2500 system (Illumina). .. We applied sequenced paired-end reads to kallisto version 0.43.1 [ ] with the “–rf-stranded -b 100” option, which estimates the transcript-level expression values as Transcripts Per Kilobase Million (TPM, Ensembl gene annotation GRCh37).

Article Title: Comparative Analysis of Impatiens Leaf Transcriptomes Reveal Candidate Genes for Resistance to Downy Mildew Caused by Plasmopara obducens
Article Snippet: .. The isolated mRNA molecules were fragmented using the Elute Prime Fragment Mix from Illumina TruSeqTM RNA sample prep kit v2 (Illumina, San Diego, CA, USA) at 94 °C for 8 min. ..

Next-Generation Sequencing:

Article Title: Co-clinical assessment identifies patterns of BRAF inhibitor resistance in melanoma
Article Snippet: .. Starting with approximately 700 ng total RNA, poly-A–containing mRNA molecules were converted into libraries suitable for next-generation sequencing approaches using reagents and the protocol provided by the Illumina TruSeq RNA Sample Prep Kit, version 2. ..

Purification:

Article Title: De Novo Sequencing and Characterization of the Floral Transcriptome of Dendrocalamus latiflorus (Poaceae: Bambusoideae)
Article Snippet: .. Poly-A mRNA molecules were purified using Sera-mag Magnetic Oligo (dT) Beads (Illumina) from 20 µg total RNA from each sample and eluted with 10 mM Tris–HCl. .. To avoid priming bias during cDNA synthesis, the purified mRNA was first fragmented into small pieces using RNA Fragmentation Reagents (Ambion, Austin, TX, USA) before cDNA synthesis.

Article Title: Genome-Wide Identification of Small RNAs in Bifidobacterium animalis subsp. lactis KLDS 2.0603 and Their Regulation Role in the Adaption to Gastrointestinal Environment
Article Snippet: .. Briefly, the poly-A containing mRNA molecules were purified using poly-T oligo-attached magnetic beads (Illumina, San Diego, CA) from total RNA, and sheared to small fragments using divalent cations under elevated temperature. ..

Article Title: Transcriptome and methylome interactions in rice hybrids
Article Snippet: .. Total RNA (10 ug) for each sample was used for purification of the poly(A)-containing mRNA molecules, RNA amplification, and synthesis of double-stranded cDNAs that were ligated to adapters, thus preparing the libraries for sequencing on the Illumina GAII. .. The RNA-seq library preparation protocol was followed as described in Illumina’s standard protocol for RNA-seq libraries (Illumina).

Sequencing:

Article Title: Transcriptome and methylome interactions in rice hybrids
Article Snippet: .. Total RNA (10 ug) for each sample was used for purification of the poly(A)-containing mRNA molecules, RNA amplification, and synthesis of double-stranded cDNAs that were ligated to adapters, thus preparing the libraries for sequencing on the Illumina GAII. .. The RNA-seq library preparation protocol was followed as described in Illumina’s standard protocol for RNA-seq libraries (Illumina).

Sample Prep:

Article Title: Co-clinical assessment identifies patterns of BRAF inhibitor resistance in melanoma
Article Snippet: .. Starting with approximately 700 ng total RNA, poly-A–containing mRNA molecules were converted into libraries suitable for next-generation sequencing approaches using reagents and the protocol provided by the Illumina TruSeq RNA Sample Prep Kit, version 2. ..

Article Title: Comprehensive epigenome characterization reveals diverse transcriptional regulation across human vascular endothelial cells
Article Snippet: .. RNA-seq analysis Poly(A)-containing mRNA molecules were isolated from total RNA and then converted to cDNA with oligo(dT) primers using a TruSeq RNA Sample Preparation kit v2 (Illumina) and were sequenced with a HiSeq 2500 system (Illumina). .. We applied sequenced paired-end reads to kallisto version 0.43.1 [ ] with the “–rf-stranded -b 100” option, which estimates the transcript-level expression values as Transcripts Per Kilobase Million (TPM, Ensembl gene annotation GRCh37).

Article Title: Comparative Analysis of Impatiens Leaf Transcriptomes Reveal Candidate Genes for Resistance to Downy Mildew Caused by Plasmopara obducens
Article Snippet: .. The isolated mRNA molecules were fragmented using the Elute Prime Fragment Mix from Illumina TruSeqTM RNA sample prep kit v2 (Illumina, San Diego, CA, USA) at 94 °C for 8 min. ..

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  • 84
    Illumina Inc vcam mrna expression
    Relationship between E‐selectin <t>mRNA</t> expression and plasma sE‐selectin levels. Association between nonalcoholic fatty liver disease (NAFLD) severity (according to NAFLD activity score) and plasma sE‐selectin levels (A; n = 58) and sVCAM levels (B; n = 58) in severely obese individuals; E‐selectin mRNA expression in liver (C; n = 62), visceral adipose tissue (VAT) (E; n = 62) and muscle (F; n = 43) in relation to plasma sE‐selectin levels. Hepatic <t>VCAM</t> mRNA expression in relation to plasma sVCAM levels (D; n = 62). Data are expressed as mean (adjusted for age and sex) ± SD, analysed with one‐way ANOVA (A and B), or as individual data points (adjusted for age and sex), analysed with linear regression (C‐F)
    Vcam Mrna Expression, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/vcam mrna expression/product/Illumina Inc
    Average 84 stars, based on 1 article reviews
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    93
    Illumina Inc transcriptome analysis
    <t>Transcriptome</t> and DAVID analyses of the HuH-7-tetOn-TSPY cells. a MA-plots showing the differentially expressed genes (DEGs) between HuH-7-tetOn-TSPY cells and HuH-7-tetOn-EGFP cells. Genes plotted within the areas of Log 2 [expression level] > 3.32 and |log 2 [fold change]| > 0.6 (red) were analyzed as differentially expressed genes (DEGs). b DAVID gene-annotation enrichment analysis identified top 5 KEGG pathways enriched in DEGs with FDR
    Transcriptome Analysis, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 93/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/transcriptome analysis/product/Illumina Inc
    Average 93 stars, based on 46 article reviews
    Price from $9.99 to $1999.99
    transcriptome analysis - by Bioz Stars, 2020-09
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    92
    Illumina Inc poly i c transfected chromatin associated rna preparations
    High-Throughput Analysis of pri-miRNA Processing during the IFN Response (A) For each pri-miRNA in <t>mock-transfected</t> cells (left) and <t>poly(I:C)-transfected</t> cells (right), the Microprocessor processing index (MPI) is calculated as shown. Log 2 fold change MPI (log 2 FC) is obtained by subtraction of MPI (mock) from MPI (poly[I:C]). (B) Less processed pri-miRNAs during the IFN response result in a positive log 2 FC MPI, in red. Equally processed pri-miRNAs in gray and more processed pri-miRNAs in green. (C) Representation of the average MPI value in mock conditions for “less processed” in red and “equally processed” pri-miRNAs in gray; ∗ p ≤ 0.05. (D) Summary of known pri-miRNA determinants of optimal Microprocessor substrates. Numbers indicate location in respect to 5′/3′ Drosha cleavage sites. (E–G) Frequency of UG (E), CNNC (F), and UGU (G) motifs for “less processed” pri-miRNAs, in red, and “equally processed,” in gray. .
    Poly I C Transfected Chromatin Associated Rna Preparations, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/poly i c transfected chromatin associated rna preparations/product/Illumina Inc
    Average 92 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
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    85
    Illumina Inc directional mrna seq library prep
    A portion of SNORD27 is present in a soluble nuclear fraction where fibrillarin is not detected. ( A ) The schematic representation of HeLa cell fractionation under native conditions. ( B ) Western blot analysis of the fractionation described in A ; 5% of the total volume of each fraction prepared was analyzed by Western blot. The components of spliceosomes (PRP8 and PRPF39), splicing regulators (CBP80/NCBP1 and hnRNPG), and constitutive SNORD binding proteins (NHP2L1/15.5K, NOP56, NOP58, and fibrillarin) were detected by specific antibodies. ( C ) Distribution of SNORD27 in cellular fractions determined by RPA. The diagram shows the SNORD27 expression cassette and the location of the RPA probe. The arrow indicates SNORD27 , and boxes indicate hosting exons. The RPA probe spanned the annotated SNORD27 sequence and 5 nt from the surrounding intron. ( D ) Separation of the soluble nuclear supernatant fraction in a glycerol gradient. Nuclear supernatant ( C ) was separated on 10–45% glycerol gradient. The gradient was subdivided into 20 fractions; <t>RNA</t> from 50% of each fraction was isolated and analyzed by RPA with an SNORD27 antisense probe. The arrow shows full-length SNORD27 . ( E ) Individual glycerol gradient fractions were analyzed by Western blotting using antibodies recognizing markers for <t>pre-mRNA</t> processing: CBP80, hnRNPG, and SRSF6. ( F ) The distribution of U1, U2 , and U4 snRNAs in the fractions obtained by HeLa cell fractionation ( A ) was determined by RPA. ( G ) Association of SNORD27 with constitutive SNORD binding proteins. Endogenous fibrillarin and Flag-tagged NHP2L1, NOP58, and NOP56 were immunoprecipitated and copurified. SNORD27 was then detected by RT-PCR. Cells transfected with GFP were used as a control. +Abs, immunoprecipitated with the depicted antibodies; H, 10 µg total HeLa RNA; M, molecular weight size marker; NP, nuclear pellet; NS, nuclear supernatant; P, untreated probe; WB, Western blot; Y, 10 µg total yeast RNA.
    Directional Mrna Seq Library Prep, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/directional mrna seq library prep/product/Illumina Inc
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    Image Search Results


    Relationship between E‐selectin mRNA expression and plasma sE‐selectin levels. Association between nonalcoholic fatty liver disease (NAFLD) severity (according to NAFLD activity score) and plasma sE‐selectin levels (A; n = 58) and sVCAM levels (B; n = 58) in severely obese individuals; E‐selectin mRNA expression in liver (C; n = 62), visceral adipose tissue (VAT) (E; n = 62) and muscle (F; n = 43) in relation to plasma sE‐selectin levels. Hepatic VCAM mRNA expression in relation to plasma sVCAM levels (D; n = 62). Data are expressed as mean (adjusted for age and sex) ± SD, analysed with one‐way ANOVA (A and B), or as individual data points (adjusted for age and sex), analysed with linear regression (C‐F)

    Journal: Liver International

    Article Title: The endothelial function biomarker soluble E‐selectin is associated with nonalcoholic fatty liver disease, et al. The endothelial function biomarker soluble E‐selectin is associated with nonalcoholic fatty liver disease

    doi: 10.1111/liv.14384

    Figure Lengend Snippet: Relationship between E‐selectin mRNA expression and plasma sE‐selectin levels. Association between nonalcoholic fatty liver disease (NAFLD) severity (according to NAFLD activity score) and plasma sE‐selectin levels (A; n = 58) and sVCAM levels (B; n = 58) in severely obese individuals; E‐selectin mRNA expression in liver (C; n = 62), visceral adipose tissue (VAT) (E; n = 62) and muscle (F; n = 43) in relation to plasma sE‐selectin levels. Hepatic VCAM mRNA expression in relation to plasma sVCAM levels (D; n = 62). Data are expressed as mean (adjusted for age and sex) ± SD, analysed with one‐way ANOVA (A and B), or as individual data points (adjusted for age and sex), analysed with linear regression (C‐F)

    Article Snippet: E‐selectin and VCAM mRNA expression in liver, VAT and muscle were determined using Illumina HumanHT12 Bead‐Chips (Illumina) and an Illumina BeadArray Reader.

    Techniques: Expressing, Activity Assay

    Transcriptome and DAVID analyses of the HuH-7-tetOn-TSPY cells. a MA-plots showing the differentially expressed genes (DEGs) between HuH-7-tetOn-TSPY cells and HuH-7-tetOn-EGFP cells. Genes plotted within the areas of Log 2 [expression level] > 3.32 and |log 2 [fold change]| > 0.6 (red) were analyzed as differentially expressed genes (DEGs). b DAVID gene-annotation enrichment analysis identified top 5 KEGG pathways enriched in DEGs with FDR

    Journal: Cell & Bioscience

    Article Title: The Y-linked proto-oncogene TSPY contributes to poor prognosis of the male hepatocellular carcinoma patients by promoting the pro-oncogenic and suppressing the anti-oncogenic gene expression

    doi: 10.1186/s13578-019-0287-x

    Figure Lengend Snippet: Transcriptome and DAVID analyses of the HuH-7-tetOn-TSPY cells. a MA-plots showing the differentially expressed genes (DEGs) between HuH-7-tetOn-TSPY cells and HuH-7-tetOn-EGFP cells. Genes plotted within the areas of Log 2 [expression level] > 3.32 and |log 2 [fold change]| > 0.6 (red) were analyzed as differentially expressed genes (DEGs). b DAVID gene-annotation enrichment analysis identified top 5 KEGG pathways enriched in DEGs with FDR

    Article Snippet: TSPY upregulated various cell-cycle related genes and suppressed tumor suppressor genes in HuH-7 cells To explore the mechanisms by which TSPY regulates the molecular events in HuH-7 cells, we had performed a transcriptome analysis using the Illumina RNA-Seq platform.

    Techniques: Expressing

    Representative expression patterns and associated with survival rates of TSPY downstream genes in HCC. a TSPY stimulated the expression levels of CDC25B, HMMR, and PPARGC1A, in HuH-7 cells. The results of transcriptome analysis (n = 3) were presented with the P-values by Student t-tests. b – d Left panels, expression levels of TSPY and selected downstream genes, e.g. CDC25B, HMMR, and PPARGC1A, among female non-tumor liver (NT), female HCC, male non-tumor liver (NT), male TSPY-silent HCC (TS(−)), and male TSPY-high HCC (TS(++)), in the TCGA transcriptome dataset. Y-axis indicates the expression level as RSEN normalized count. Right panels, survival analyses of the TCGA data, comparing the high-expresser HCC patients (red lines) and the low-expresser HCC patients (blue lines) for the respective genes. Both male and female cases were included in these analyses. Log-rank test P-values are indicated. Abbreviations; **t-test P-value

    Journal: Cell & Bioscience

    Article Title: The Y-linked proto-oncogene TSPY contributes to poor prognosis of the male hepatocellular carcinoma patients by promoting the pro-oncogenic and suppressing the anti-oncogenic gene expression

    doi: 10.1186/s13578-019-0287-x

    Figure Lengend Snippet: Representative expression patterns and associated with survival rates of TSPY downstream genes in HCC. a TSPY stimulated the expression levels of CDC25B, HMMR, and PPARGC1A, in HuH-7 cells. The results of transcriptome analysis (n = 3) were presented with the P-values by Student t-tests. b – d Left panels, expression levels of TSPY and selected downstream genes, e.g. CDC25B, HMMR, and PPARGC1A, among female non-tumor liver (NT), female HCC, male non-tumor liver (NT), male TSPY-silent HCC (TS(−)), and male TSPY-high HCC (TS(++)), in the TCGA transcriptome dataset. Y-axis indicates the expression level as RSEN normalized count. Right panels, survival analyses of the TCGA data, comparing the high-expresser HCC patients (red lines) and the low-expresser HCC patients (blue lines) for the respective genes. Both male and female cases were included in these analyses. Log-rank test P-values are indicated. Abbreviations; **t-test P-value

    Article Snippet: TSPY upregulated various cell-cycle related genes and suppressed tumor suppressor genes in HuH-7 cells To explore the mechanisms by which TSPY regulates the molecular events in HuH-7 cells, we had performed a transcriptome analysis using the Illumina RNA-Seq platform.

    Techniques: Expressing

    High-Throughput Analysis of pri-miRNA Processing during the IFN Response (A) For each pri-miRNA in mock-transfected cells (left) and poly(I:C)-transfected cells (right), the Microprocessor processing index (MPI) is calculated as shown. Log 2 fold change MPI (log 2 FC) is obtained by subtraction of MPI (mock) from MPI (poly[I:C]). (B) Less processed pri-miRNAs during the IFN response result in a positive log 2 FC MPI, in red. Equally processed pri-miRNAs in gray and more processed pri-miRNAs in green. (C) Representation of the average MPI value in mock conditions for “less processed” in red and “equally processed” pri-miRNAs in gray; ∗ p ≤ 0.05. (D) Summary of known pri-miRNA determinants of optimal Microprocessor substrates. Numbers indicate location in respect to 5′/3′ Drosha cleavage sites. (E–G) Frequency of UG (E), CNNC (F), and UGU (G) motifs for “less processed” pri-miRNAs, in red, and “equally processed,” in gray. .

    Journal: Cell Reports

    Article Title: Inhibition of Microprocessor Function during the Activation of the Type I Interferon Response

    doi: 10.1016/j.celrep.2018.05.049

    Figure Lengend Snippet: High-Throughput Analysis of pri-miRNA Processing during the IFN Response (A) For each pri-miRNA in mock-transfected cells (left) and poly(I:C)-transfected cells (right), the Microprocessor processing index (MPI) is calculated as shown. Log 2 fold change MPI (log 2 FC) is obtained by subtraction of MPI (mock) from MPI (poly[I:C]). (B) Less processed pri-miRNAs during the IFN response result in a positive log 2 FC MPI, in red. Equally processed pri-miRNAs in gray and more processed pri-miRNAs in green. (C) Representation of the average MPI value in mock conditions for “less processed” in red and “equally processed” pri-miRNAs in gray; ∗ p ≤ 0.05. (D) Summary of known pri-miRNA determinants of optimal Microprocessor substrates. Numbers indicate location in respect to 5′/3′ Drosha cleavage sites. (E–G) Frequency of UG (E), CNNC (F), and UGU (G) motifs for “less processed” pri-miRNAs, in red, and “equally processed,” in gray. .

    Article Snippet: Four mock-transfected and four poly(I:C)-transfected chromatin-associated RNA preparations were generated for strand-specific RNA transcriptome sequencing, including ribo-zero rRNA depletion and random fragmentation and strand-specific library construction and sequenced by Illumina HiSeq 4000, 100PE.

    Techniques: High Throughput Screening Assay, Transfection

    A portion of SNORD27 is present in a soluble nuclear fraction where fibrillarin is not detected. ( A ) The schematic representation of HeLa cell fractionation under native conditions. ( B ) Western blot analysis of the fractionation described in A ; 5% of the total volume of each fraction prepared was analyzed by Western blot. The components of spliceosomes (PRP8 and PRPF39), splicing regulators (CBP80/NCBP1 and hnRNPG), and constitutive SNORD binding proteins (NHP2L1/15.5K, NOP56, NOP58, and fibrillarin) were detected by specific antibodies. ( C ) Distribution of SNORD27 in cellular fractions determined by RPA. The diagram shows the SNORD27 expression cassette and the location of the RPA probe. The arrow indicates SNORD27 , and boxes indicate hosting exons. The RPA probe spanned the annotated SNORD27 sequence and 5 nt from the surrounding intron. ( D ) Separation of the soluble nuclear supernatant fraction in a glycerol gradient. Nuclear supernatant ( C ) was separated on 10–45% glycerol gradient. The gradient was subdivided into 20 fractions; RNA from 50% of each fraction was isolated and analyzed by RPA with an SNORD27 antisense probe. The arrow shows full-length SNORD27 . ( E ) Individual glycerol gradient fractions were analyzed by Western blotting using antibodies recognizing markers for pre-mRNA processing: CBP80, hnRNPG, and SRSF6. ( F ) The distribution of U1, U2 , and U4 snRNAs in the fractions obtained by HeLa cell fractionation ( A ) was determined by RPA. ( G ) Association of SNORD27 with constitutive SNORD binding proteins. Endogenous fibrillarin and Flag-tagged NHP2L1, NOP58, and NOP56 were immunoprecipitated and copurified. SNORD27 was then detected by RT-PCR. Cells transfected with GFP were used as a control. +Abs, immunoprecipitated with the depicted antibodies; H, 10 µg total HeLa RNA; M, molecular weight size marker; NP, nuclear pellet; NS, nuclear supernatant; P, untreated probe; WB, Western blot; Y, 10 µg total yeast RNA.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Dual function of C/D box small nucleolar RNAs in rRNA modification and alternative pre-mRNA splicing

    doi: 10.1073/pnas.1519292113

    Figure Lengend Snippet: A portion of SNORD27 is present in a soluble nuclear fraction where fibrillarin is not detected. ( A ) The schematic representation of HeLa cell fractionation under native conditions. ( B ) Western blot analysis of the fractionation described in A ; 5% of the total volume of each fraction prepared was analyzed by Western blot. The components of spliceosomes (PRP8 and PRPF39), splicing regulators (CBP80/NCBP1 and hnRNPG), and constitutive SNORD binding proteins (NHP2L1/15.5K, NOP56, NOP58, and fibrillarin) were detected by specific antibodies. ( C ) Distribution of SNORD27 in cellular fractions determined by RPA. The diagram shows the SNORD27 expression cassette and the location of the RPA probe. The arrow indicates SNORD27 , and boxes indicate hosting exons. The RPA probe spanned the annotated SNORD27 sequence and 5 nt from the surrounding intron. ( D ) Separation of the soluble nuclear supernatant fraction in a glycerol gradient. Nuclear supernatant ( C ) was separated on 10–45% glycerol gradient. The gradient was subdivided into 20 fractions; RNA from 50% of each fraction was isolated and analyzed by RPA with an SNORD27 antisense probe. The arrow shows full-length SNORD27 . ( E ) Individual glycerol gradient fractions were analyzed by Western blotting using antibodies recognizing markers for pre-mRNA processing: CBP80, hnRNPG, and SRSF6. ( F ) The distribution of U1, U2 , and U4 snRNAs in the fractions obtained by HeLa cell fractionation ( A ) was determined by RPA. ( G ) Association of SNORD27 with constitutive SNORD binding proteins. Endogenous fibrillarin and Flag-tagged NHP2L1, NOP58, and NOP56 were immunoprecipitated and copurified. SNORD27 was then detected by RT-PCR. Cells transfected with GFP were used as a control. +Abs, immunoprecipitated with the depicted antibodies; H, 10 µg total HeLa RNA; M, molecular weight size marker; NP, nuclear pellet; NS, nuclear supernatant; P, untreated probe; WB, Western blot; Y, 10 µg total yeast RNA.

    Article Snippet: For small RNA library construction, ∼10 µg RNA was used followed by Illumina Directional mRNA-Seq Library Prep.

    Techniques: Cell Fractionation, Western Blot, Fractionation, Binding Assay, Recombinase Polymerase Amplification, Expressing, Sequencing, Isolation, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Transfection, Molecular Weight, Marker

    SNORD27 binds to E2F7 pre-mRNA in vivo. ( A ) Schematic representation of the E2F7-PP7 minigene with a PP7 RNA tag at the 5′ end. The location of the SNORD27 binding site and amplification primers are indicated. ( B ) Purification scheme. Soluble nuclear fractions were obtained from HeLa cells transfected with PP7-tagged E2F7 minigene and incubated with PP7 binding protein fused to protein A. Complexes were isolated using IgG-coated beads and eluted using TEV-protease cleavage. The diagram does not depict the spliceosomal complex within which the eluted transcript is associated. ( C ) RT-PCR of RNAs eluted after PP7 tag purification. E2F7 and SNORD27 were amplified by RT-PCR, and SNORD53 and β-actin were used as negative controls. *Location of an unspecific extra band. ( D ) Western blot showing the elution profile of the spliceosome-associated protein PRPF39 . Lanes correspond to those in C .

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Dual function of C/D box small nucleolar RNAs in rRNA modification and alternative pre-mRNA splicing

    doi: 10.1073/pnas.1519292113

    Figure Lengend Snippet: SNORD27 binds to E2F7 pre-mRNA in vivo. ( A ) Schematic representation of the E2F7-PP7 minigene with a PP7 RNA tag at the 5′ end. The location of the SNORD27 binding site and amplification primers are indicated. ( B ) Purification scheme. Soluble nuclear fractions were obtained from HeLa cells transfected with PP7-tagged E2F7 minigene and incubated with PP7 binding protein fused to protein A. Complexes were isolated using IgG-coated beads and eluted using TEV-protease cleavage. The diagram does not depict the spliceosomal complex within which the eluted transcript is associated. ( C ) RT-PCR of RNAs eluted after PP7 tag purification. E2F7 and SNORD27 were amplified by RT-PCR, and SNORD53 and β-actin were used as negative controls. *Location of an unspecific extra band. ( D ) Western blot showing the elution profile of the spliceosome-associated protein PRPF39 . Lanes correspond to those in C .

    Article Snippet: For small RNA library construction, ∼10 µg RNA was used followed by Illumina Directional mRNA-Seq Library Prep.

    Techniques: In Vivo, Binding Assay, Amplification, Purification, Transfection, Incubation, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot