glycoblue co-precipitant Thermo Fisher Search Results


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  • 99
    Thermo Fisher glycoblue coprecipitant
    eIF4F disassembly does not deplete viral mRNAs from polysomes. (A) Polysome profile of TRex-BCBLl-RTA cells induced with 1 µg/mL dox for 24 h. Torin or DMSO control were added 2 h prior to harvest and polysome analysis. (B) qRT-PCR analysis of cellular and viral transcripts in polysome fractions. Total RNA was isolated from polysome fractions. RNA was co-precipitated with <t>GlycoBlue</t> and T7-transcribed luciferase RNA to improve and normalize for recovery. RNA was analysed by qRT-PCR for cellular, and viral transcripts. Vertical lines depict the boundaries between the monosomes, light polysome, and heavy polysome fractions (n= 3; means±SEM; statistical significance was determined by two-way ANOVA).
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    Thermo Fisher trizol
    eIF4F disassembly does not deplete viral mRNAs from polysomes. (A) Polysome profile of TRex-BCBLl-RTA cells induced with 1 µg/mL dox for 24 h. Torin or DMSO control were added 2 h prior to harvest and polysome analysis. (B) qRT-PCR analysis of cellular and viral transcripts in polysome fractions. Total RNA was isolated from polysome fractions. RNA was co-precipitated with <t>GlycoBlue</t> and T7-transcribed luciferase RNA to improve and normalize for recovery. RNA was analysed by qRT-PCR for cellular, and viral transcripts. Vertical lines depict the boundaries between the monosomes, light polysome, and heavy polysome fractions (n= 3; means±SEM; statistical significance was determined by two-way ANOVA).
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    Thermo Fisher sodium acetate
    eIF4F disassembly does not deplete viral mRNAs from polysomes. (A) Polysome profile of TRex-BCBLl-RTA cells induced with 1 µg/mL dox for 24 h. Torin or DMSO control were added 2 h prior to harvest and polysome analysis. (B) qRT-PCR analysis of cellular and viral transcripts in polysome fractions. Total RNA was isolated from polysome fractions. RNA was co-precipitated with <t>GlycoBlue</t> and T7-transcribed luciferase RNA to improve and normalize for recovery. RNA was analysed by qRT-PCR for cellular, and viral transcripts. Vertical lines depict the boundaries between the monosomes, light polysome, and heavy polysome fractions (n= 3; means±SEM; statistical significance was determined by two-way ANOVA).
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    Thermo Fisher total rna
    Quantitative Detection of Internal m 7 G Sites in Mammalian Messenger <t>RNA</t> and MeRIP-seq Profile in Mammalian Cells. (A) Chemical structure of N 7 -methylguanosine (m 7 G). (B) LC-MS/MS quantification of m 7 G/G levels in polyA + RNA (HEK293T cells) using a three-step RNA digestion (red) or a two-step digestion (blue). Mean values ± s.d. are shown, n = 3. (C) LC-MS/MS quantification of m 7 G/G and m 6 A/A levels in cap-depleted polyA + <t>mRNA</t> isolated from human and mouse cells. Mean values ± s.d. are shown, n = 3. (D) LC-MS/MS quantification of m 7 G/G, Gm/G, m 6 A/A, Am/A, m 1 A/A levels showing enrichment of m 7 G after immunoprecipitation using anti-m 7 G specific antibody (MBL), without enriching other modifications. Cap-depleted polyA + mRNA (HEK293T cells) was used as the input control. Mean values ± s.d. are shown, n = 3. (E) The overlap of m 7 G-MeRIP-seq m 7 G peaks among three human cell lines (left) and two mouse cell lines (right) (fold change (FC) ≥ 3, false discovery rate (FDR) ≤ 0.05, FPKM ≥ 1.0). (F) The percentage of methylated genes with 1, 2, 3, 4 or 5+ peaks per gene in the indicated human and mouse cell types. (G) Top three motifs identified with m 7 G-MeRIP-seq by HOMER software.
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    Thermo Fisher nuclease free water
    Quantitative Detection of Internal m 7 G Sites in Mammalian Messenger <t>RNA</t> and MeRIP-seq Profile in Mammalian Cells. (A) Chemical structure of N 7 -methylguanosine (m 7 G). (B) LC-MS/MS quantification of m 7 G/G levels in polyA + RNA (HEK293T cells) using a three-step RNA digestion (red) or a two-step digestion (blue). Mean values ± s.d. are shown, n = 3. (C) LC-MS/MS quantification of m 7 G/G and m 6 A/A levels in cap-depleted polyA + <t>mRNA</t> isolated from human and mouse cells. Mean values ± s.d. are shown, n = 3. (D) LC-MS/MS quantification of m 7 G/G, Gm/G, m 6 A/A, Am/A, m 1 A/A levels showing enrichment of m 7 G after immunoprecipitation using anti-m 7 G specific antibody (MBL), without enriching other modifications. Cap-depleted polyA + mRNA (HEK293T cells) was used as the input control. Mean values ± s.d. are shown, n = 3. (E) The overlap of m 7 G-MeRIP-seq m 7 G peaks among three human cell lines (left) and two mouse cell lines (right) (fold change (FC) ≥ 3, false discovery rate (FDR) ≤ 0.05, FPKM ≥ 1.0). (F) The percentage of methylated genes with 1, 2, 3, 4 or 5+ peaks per gene in the indicated human and mouse cell types. (G) Top three motifs identified with m 7 G-MeRIP-seq by HOMER software.
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    Thermo Fisher acid phenol chloroform
    Quantitative Detection of Internal m 7 G Sites in Mammalian Messenger <t>RNA</t> and MeRIP-seq Profile in Mammalian Cells. (A) Chemical structure of N 7 -methylguanosine (m 7 G). (B) LC-MS/MS quantification of m 7 G/G levels in polyA + RNA (HEK293T cells) using a three-step RNA digestion (red) or a two-step digestion (blue). Mean values ± s.d. are shown, n = 3. (C) LC-MS/MS quantification of m 7 G/G and m 6 A/A levels in cap-depleted polyA + <t>mRNA</t> isolated from human and mouse cells. Mean values ± s.d. are shown, n = 3. (D) LC-MS/MS quantification of m 7 G/G, Gm/G, m 6 A/A, Am/A, m 1 A/A levels showing enrichment of m 7 G after immunoprecipitation using anti-m 7 G specific antibody (MBL), without enriching other modifications. Cap-depleted polyA + mRNA (HEK293T cells) was used as the input control. Mean values ± s.d. are shown, n = 3. (E) The overlap of m 7 G-MeRIP-seq m 7 G peaks among three human cell lines (left) and two mouse cell lines (right) (fold change (FC) ≥ 3, false discovery rate (FDR) ≤ 0.05, FPKM ≥ 1.0). (F) The percentage of methylated genes with 1, 2, 3, 4 or 5+ peaks per gene in the indicated human and mouse cell types. (G) Top three motifs identified with m 7 G-MeRIP-seq by HOMER software.
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    Thermo Fisher co precipitant
    Quantitative Detection of Internal m 7 G Sites in Mammalian Messenger <t>RNA</t> and MeRIP-seq Profile in Mammalian Cells. (A) Chemical structure of N 7 -methylguanosine (m 7 G). (B) LC-MS/MS quantification of m 7 G/G levels in polyA + RNA (HEK293T cells) using a three-step RNA digestion (red) or a two-step digestion (blue). Mean values ± s.d. are shown, n = 3. (C) LC-MS/MS quantification of m 7 G/G and m 6 A/A levels in cap-depleted polyA + <t>mRNA</t> isolated from human and mouse cells. Mean values ± s.d. are shown, n = 3. (D) LC-MS/MS quantification of m 7 G/G, Gm/G, m 6 A/A, Am/A, m 1 A/A levels showing enrichment of m 7 G after immunoprecipitation using anti-m 7 G specific antibody (MBL), without enriching other modifications. Cap-depleted polyA + mRNA (HEK293T cells) was used as the input control. Mean values ± s.d. are shown, n = 3. (E) The overlap of m 7 G-MeRIP-seq m 7 G peaks among three human cell lines (left) and two mouse cell lines (right) (fold change (FC) ≥ 3, false discovery rate (FDR) ≤ 0.05, FPKM ≥ 1.0). (F) The percentage of methylated genes with 1, 2, 3, 4 or 5+ peaks per gene in the indicated human and mouse cell types. (G) Top three motifs identified with m 7 G-MeRIP-seq by HOMER software.
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    Thermo Fisher dnase i
    Quantitative Detection of Internal m 7 G Sites in Mammalian Messenger <t>RNA</t> and MeRIP-seq Profile in Mammalian Cells. (A) Chemical structure of N 7 -methylguanosine (m 7 G). (B) LC-MS/MS quantification of m 7 G/G levels in polyA + RNA (HEK293T cells) using a three-step RNA digestion (red) or a two-step digestion (blue). Mean values ± s.d. are shown, n = 3. (C) LC-MS/MS quantification of m 7 G/G and m 6 A/A levels in cap-depleted polyA + <t>mRNA</t> isolated from human and mouse cells. Mean values ± s.d. are shown, n = 3. (D) LC-MS/MS quantification of m 7 G/G, Gm/G, m 6 A/A, Am/A, m 1 A/A levels showing enrichment of m 7 G after immunoprecipitation using anti-m 7 G specific antibody (MBL), without enriching other modifications. Cap-depleted polyA + mRNA (HEK293T cells) was used as the input control. Mean values ± s.d. are shown, n = 3. (E) The overlap of m 7 G-MeRIP-seq m 7 G peaks among three human cell lines (left) and two mouse cell lines (right) (fold change (FC) ≥ 3, false discovery rate (FDR) ≤ 0.05, FPKM ≥ 1.0). (F) The percentage of methylated genes with 1, 2, 3, 4 or 5+ peaks per gene in the indicated human and mouse cell types. (G) Top three motifs identified with m 7 G-MeRIP-seq by HOMER software.
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    Thermo Fisher nacl
    Quantitative Detection of Internal m 7 G Sites in Mammalian Messenger <t>RNA</t> and MeRIP-seq Profile in Mammalian Cells. (A) Chemical structure of N 7 -methylguanosine (m 7 G). (B) LC-MS/MS quantification of m 7 G/G levels in polyA + RNA (HEK293T cells) using a three-step RNA digestion (red) or a two-step digestion (blue). Mean values ± s.d. are shown, n = 3. (C) LC-MS/MS quantification of m 7 G/G and m 6 A/A levels in cap-depleted polyA + <t>mRNA</t> isolated from human and mouse cells. Mean values ± s.d. are shown, n = 3. (D) LC-MS/MS quantification of m 7 G/G, Gm/G, m 6 A/A, Am/A, m 1 A/A levels showing enrichment of m 7 G after immunoprecipitation using anti-m 7 G specific antibody (MBL), without enriching other modifications. Cap-depleted polyA + mRNA (HEK293T cells) was used as the input control. Mean values ± s.d. are shown, n = 3. (E) The overlap of m 7 G-MeRIP-seq m 7 G peaks among three human cell lines (left) and two mouse cell lines (right) (fold change (FC) ≥ 3, false discovery rate (FDR) ≤ 0.05, FPKM ≥ 1.0). (F) The percentage of methylated genes with 1, 2, 3, 4 or 5+ peaks per gene in the indicated human and mouse cell types. (G) Top three motifs identified with m 7 G-MeRIP-seq by HOMER software.
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    Thermo Fisher trizol ls reagent
    Quantitative Detection of Internal m 7 G Sites in Mammalian Messenger <t>RNA</t> and MeRIP-seq Profile in Mammalian Cells. (A) Chemical structure of N 7 -methylguanosine (m 7 G). (B) LC-MS/MS quantification of m 7 G/G levels in polyA + RNA (HEK293T cells) using a three-step RNA digestion (red) or a two-step digestion (blue). Mean values ± s.d. are shown, n = 3. (C) LC-MS/MS quantification of m 7 G/G and m 6 A/A levels in cap-depleted polyA + <t>mRNA</t> isolated from human and mouse cells. Mean values ± s.d. are shown, n = 3. (D) LC-MS/MS quantification of m 7 G/G, Gm/G, m 6 A/A, Am/A, m 1 A/A levels showing enrichment of m 7 G after immunoprecipitation using anti-m 7 G specific antibody (MBL), without enriching other modifications. Cap-depleted polyA + mRNA (HEK293T cells) was used as the input control. Mean values ± s.d. are shown, n = 3. (E) The overlap of m 7 G-MeRIP-seq m 7 G peaks among three human cell lines (left) and two mouse cell lines (right) (fold change (FC) ≥ 3, false discovery rate (FDR) ≤ 0.05, FPKM ≥ 1.0). (F) The percentage of methylated genes with 1, 2, 3, 4 or 5+ peaks per gene in the indicated human and mouse cell types. (G) Top three motifs identified with m 7 G-MeRIP-seq by HOMER software.
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    Thermo Fisher proteinase k
    Quantitative Detection of Internal m 7 G Sites in Mammalian Messenger <t>RNA</t> and MeRIP-seq Profile in Mammalian Cells. (A) Chemical structure of N 7 -methylguanosine (m 7 G). (B) LC-MS/MS quantification of m 7 G/G levels in polyA + RNA (HEK293T cells) using a three-step RNA digestion (red) or a two-step digestion (blue). Mean values ± s.d. are shown, n = 3. (C) LC-MS/MS quantification of m 7 G/G and m 6 A/A levels in cap-depleted polyA + <t>mRNA</t> isolated from human and mouse cells. Mean values ± s.d. are shown, n = 3. (D) LC-MS/MS quantification of m 7 G/G, Gm/G, m 6 A/A, Am/A, m 1 A/A levels showing enrichment of m 7 G after immunoprecipitation using anti-m 7 G specific antibody (MBL), without enriching other modifications. Cap-depleted polyA + mRNA (HEK293T cells) was used as the input control. Mean values ± s.d. are shown, n = 3. (E) The overlap of m 7 G-MeRIP-seq m 7 G peaks among three human cell lines (left) and two mouse cell lines (right) (fold change (FC) ≥ 3, false discovery rate (FDR) ≤ 0.05, FPKM ≥ 1.0). (F) The percentage of methylated genes with 1, 2, 3, 4 or 5+ peaks per gene in the indicated human and mouse cell types. (G) Top three motifs identified with m 7 G-MeRIP-seq by HOMER software.
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    Thermo Fisher superscript iii reverse transcriptase
    Characterization of the association between Gag and HIV-1 <t>RNA</t> . (A) Measurement of the association between Gag and HIV-1 genomic RNA. HIV-1 genomic RNA immunoprecipitated with the Gag was characterized by qPCR using a primer/probe set targeting the unspliced RNA transcript. (B) Measurement of the association between Gag and spliced HIV-1 mRNA. The same cDNA preparation described above was subjected to qPCR using a primer/probe set specific for env mRNA and rev mRNA sequences. The copy number in each sample was adjusted for input by the cell number and for transfection efficiency by GFP expression from a co-transfected reporter construct. The amount of NL4-3 RNA was set at 100%. Means and SD of <t>three</t> independent experiments are shown. *, indicates p
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    Thermo Fisher phenol chloroform isoamyl alcohol
    Characterization of the association between Gag and HIV-1 <t>RNA</t> . (A) Measurement of the association between Gag and HIV-1 genomic RNA. HIV-1 genomic RNA immunoprecipitated with the Gag was characterized by qPCR using a primer/probe set targeting the unspliced RNA transcript. (B) Measurement of the association between Gag and spliced HIV-1 mRNA. The same cDNA preparation described above was subjected to qPCR using a primer/probe set specific for env mRNA and rev mRNA sequences. The copy number in each sample was adjusted for input by the cell number and for transfection efficiency by GFP expression from a co-transfected reporter construct. The amount of NL4-3 RNA was set at 100%. Means and SD of <t>three</t> independent experiments are shown. *, indicates p
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    Fisher Scientific serological pipettes
    Characterization of the association between Gag and HIV-1 <t>RNA</t> . (A) Measurement of the association between Gag and HIV-1 genomic RNA. HIV-1 genomic RNA immunoprecipitated with the Gag was characterized by qPCR using a primer/probe set targeting the unspliced RNA transcript. (B) Measurement of the association between Gag and spliced HIV-1 mRNA. The same cDNA preparation described above was subjected to qPCR using a primer/probe set specific for env mRNA and rev mRNA sequences. The copy number in each sample was adjusted for input by the cell number and for transfection efficiency by GFP expression from a co-transfected reporter construct. The amount of NL4-3 RNA was set at 100%. Means and SD of <t>three</t> independent experiments are shown. *, indicates p
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    Agilent technologies 2100 bioanalyzer
    Characterization of the association between Gag and HIV-1 <t>RNA</t> . (A) Measurement of the association between Gag and HIV-1 genomic RNA. HIV-1 genomic RNA immunoprecipitated with the Gag was characterized by qPCR using a primer/probe set targeting the unspliced RNA transcript. (B) Measurement of the association between Gag and spliced HIV-1 mRNA. The same cDNA preparation described above was subjected to qPCR using a primer/probe set specific for env mRNA and rev mRNA sequences. The copy number in each sample was adjusted for input by the cell number and for transfection efficiency by GFP expression from a co-transfected reporter construct. The amount of NL4-3 RNA was set at 100%. Means and SD of <t>three</t> independent experiments are shown. *, indicates p
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    Characterization of the association between Gag and HIV-1 <t>RNA</t> . (A) Measurement of the association between Gag and HIV-1 genomic RNA. HIV-1 genomic RNA immunoprecipitated with the Gag was characterized by qPCR using a primer/probe set targeting the unspliced RNA transcript. (B) Measurement of the association between Gag and spliced HIV-1 mRNA. The same cDNA preparation described above was subjected to qPCR using a primer/probe set specific for env mRNA and rev mRNA sequences. The copy number in each sample was adjusted for input by the cell number and for transfection efficiency by GFP expression from a co-transfected reporter construct. The amount of NL4-3 RNA was set at 100%. Means and SD of <t>three</t> independent experiments are shown. *, indicates p
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    Characterization of the association between Gag and HIV-1 <t>RNA</t> . (A) Measurement of the association between Gag and HIV-1 genomic RNA. HIV-1 genomic RNA immunoprecipitated with the Gag was characterized by qPCR using a primer/probe set targeting the unspliced RNA transcript. (B) Measurement of the association between Gag and spliced HIV-1 mRNA. The same cDNA preparation described above was subjected to qPCR using a primer/probe set specific for env mRNA and rev mRNA sequences. The copy number in each sample was adjusted for input by the cell number and for transfection efficiency by GFP expression from a co-transfected reporter construct. The amount of NL4-3 RNA was set at 100%. Means and SD of <t>three</t> independent experiments are shown. *, indicates p
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    Image Search Results


    eIF4F disassembly does not deplete viral mRNAs from polysomes. (A) Polysome profile of TRex-BCBLl-RTA cells induced with 1 µg/mL dox for 24 h. Torin or DMSO control were added 2 h prior to harvest and polysome analysis. (B) qRT-PCR analysis of cellular and viral transcripts in polysome fractions. Total RNA was isolated from polysome fractions. RNA was co-precipitated with GlycoBlue and T7-transcribed luciferase RNA to improve and normalize for recovery. RNA was analysed by qRT-PCR for cellular, and viral transcripts. Vertical lines depict the boundaries between the monosomes, light polysome, and heavy polysome fractions (n= 3; means±SEM; statistical significance was determined by two-way ANOVA).

    Journal: bioRxiv

    Article Title: KSHV lytic mRNA is efficiently translated in the absence of eIF4F

    doi: 10.1101/356162

    Figure Lengend Snippet: eIF4F disassembly does not deplete viral mRNAs from polysomes. (A) Polysome profile of TRex-BCBLl-RTA cells induced with 1 µg/mL dox for 24 h. Torin or DMSO control were added 2 h prior to harvest and polysome analysis. (B) qRT-PCR analysis of cellular and viral transcripts in polysome fractions. Total RNA was isolated from polysome fractions. RNA was co-precipitated with GlycoBlue and T7-transcribed luciferase RNA to improve and normalize for recovery. RNA was analysed by qRT-PCR for cellular, and viral transcripts. Vertical lines depict the boundaries between the monosomes, light polysome, and heavy polysome fractions (n= 3; means±SEM; statistical significance was determined by two-way ANOVA).

    Article Snippet: Fractions were mixed 1:1 with Trizol and isolated as per manufacturer’s directions except that 30-60 µg of GlycoBlue Co-Precipitant (Ambion) and 100 ng of in vitro transcribed luciferase DNA (NEB T7 HiScribe) was added to the aqueous fractions during isopropanol precipitation ( ).

    Techniques: Quantitative RT-PCR, Isolation, Luciferase

    Quantitative Detection of Internal m 7 G Sites in Mammalian Messenger RNA and MeRIP-seq Profile in Mammalian Cells. (A) Chemical structure of N 7 -methylguanosine (m 7 G). (B) LC-MS/MS quantification of m 7 G/G levels in polyA + RNA (HEK293T cells) using a three-step RNA digestion (red) or a two-step digestion (blue). Mean values ± s.d. are shown, n = 3. (C) LC-MS/MS quantification of m 7 G/G and m 6 A/A levels in cap-depleted polyA + mRNA isolated from human and mouse cells. Mean values ± s.d. are shown, n = 3. (D) LC-MS/MS quantification of m 7 G/G, Gm/G, m 6 A/A, Am/A, m 1 A/A levels showing enrichment of m 7 G after immunoprecipitation using anti-m 7 G specific antibody (MBL), without enriching other modifications. Cap-depleted polyA + mRNA (HEK293T cells) was used as the input control. Mean values ± s.d. are shown, n = 3. (E) The overlap of m 7 G-MeRIP-seq m 7 G peaks among three human cell lines (left) and two mouse cell lines (right) (fold change (FC) ≥ 3, false discovery rate (FDR) ≤ 0.05, FPKM ≥ 1.0). (F) The percentage of methylated genes with 1, 2, 3, 4 or 5+ peaks per gene in the indicated human and mouse cell types. (G) Top three motifs identified with m 7 G-MeRIP-seq by HOMER software.

    Journal: Molecular cell

    Article Title: Transcriptome-wide Mapping of Internal N7-methylguanosine Methylome in Mammalian Messenger RNA

    doi: 10.1016/j.molcel.2019.03.036

    Figure Lengend Snippet: Quantitative Detection of Internal m 7 G Sites in Mammalian Messenger RNA and MeRIP-seq Profile in Mammalian Cells. (A) Chemical structure of N 7 -methylguanosine (m 7 G). (B) LC-MS/MS quantification of m 7 G/G levels in polyA + RNA (HEK293T cells) using a three-step RNA digestion (red) or a two-step digestion (blue). Mean values ± s.d. are shown, n = 3. (C) LC-MS/MS quantification of m 7 G/G and m 6 A/A levels in cap-depleted polyA + mRNA isolated from human and mouse cells. Mean values ± s.d. are shown, n = 3. (D) LC-MS/MS quantification of m 7 G/G, Gm/G, m 6 A/A, Am/A, m 1 A/A levels showing enrichment of m 7 G after immunoprecipitation using anti-m 7 G specific antibody (MBL), without enriching other modifications. Cap-depleted polyA + mRNA (HEK293T cells) was used as the input control. Mean values ± s.d. are shown, n = 3. (E) The overlap of m 7 G-MeRIP-seq m 7 G peaks among three human cell lines (left) and two mouse cell lines (right) (fold change (FC) ≥ 3, false discovery rate (FDR) ≤ 0.05, FPKM ≥ 1.0). (F) The percentage of methylated genes with 1, 2, 3, 4 or 5+ peaks per gene in the indicated human and mouse cell types. (G) Top three motifs identified with m 7 G-MeRIP-seq by HOMER software.

    Article Snippet: Total RNA isolation for RT-qPCR: total RNA was isolated from wild-type or transiently transfected cells with TRIzol reagent (Invitrogen) and then extracted with Direct-zol RNA MiniPrep (Zymo Research), including an on-column DNase I digestion. mRNA isolation for LC-MS/MS: total RNA was extracted as described previously. mRNA was extracted by two rounds of polyA+ purification with Dynabeads mRNA DIRECT kit (Ambion). mRNA concentration was measured using Qubit RNA HS Assay Kit (Thermo Fisher Scientific) with Qubit 2.0 fluorometer.

    Techniques: Liquid Chromatography with Mass Spectroscopy, Isolation, Immunoprecipitation, Methylation, Software

    METTL1 Mediates Internal mRNA m 7 G Methylation. (A) Pie chart showing the fraction of hypo-methylated m 7 G peaks from m 7 G-MeRIP-seq of siMETTL1 HeLa and shMETTL1 HepG2 cells in each of three non-overlapping transcript segments. (B) Top two motifs identified from hypo-methylated m 7 G peaks for siMETTL1 HeLa cells and shMETTL1 HepG2 cells. (C) A global reduction of m 7 G-meRIP-seq intensity was observed within hypo-methylated peaks upon METTL1 knockdown. Two sided Mann-Whitney test. (D) GO analysis for potential METTL1 target transcripts that contains hypo-methylated peaks in siMETTL1 HeLa cells. (E) Left (light blue): LC-MS/MS results of internal m 7 G/G levels in HepG2 small RNA (

    Journal: Molecular cell

    Article Title: Transcriptome-wide Mapping of Internal N7-methylguanosine Methylome in Mammalian Messenger RNA

    doi: 10.1016/j.molcel.2019.03.036

    Figure Lengend Snippet: METTL1 Mediates Internal mRNA m 7 G Methylation. (A) Pie chart showing the fraction of hypo-methylated m 7 G peaks from m 7 G-MeRIP-seq of siMETTL1 HeLa and shMETTL1 HepG2 cells in each of three non-overlapping transcript segments. (B) Top two motifs identified from hypo-methylated m 7 G peaks for siMETTL1 HeLa cells and shMETTL1 HepG2 cells. (C) A global reduction of m 7 G-meRIP-seq intensity was observed within hypo-methylated peaks upon METTL1 knockdown. Two sided Mann-Whitney test. (D) GO analysis for potential METTL1 target transcripts that contains hypo-methylated peaks in siMETTL1 HeLa cells. (E) Left (light blue): LC-MS/MS results of internal m 7 G/G levels in HepG2 small RNA (

    Article Snippet: Total RNA isolation for RT-qPCR: total RNA was isolated from wild-type or transiently transfected cells with TRIzol reagent (Invitrogen) and then extracted with Direct-zol RNA MiniPrep (Zymo Research), including an on-column DNase I digestion. mRNA isolation for LC-MS/MS: total RNA was extracted as described previously. mRNA was extracted by two rounds of polyA+ purification with Dynabeads mRNA DIRECT kit (Ambion). mRNA concentration was measured using Qubit RNA HS Assay Kit (Thermo Fisher Scientific) with Qubit 2.0 fluorometer.

    Techniques: Methylation, MANN-WHITNEY, Liquid Chromatography with Mass Spectroscopy

    ) were transfected with expression constructs encoding each noted transcription factor linked by an IRES to a GFP reporter open reading frame (see “Materials and Methods”). Transfected cells were isolated by GFP FACS and their RNA content was assayed for specific marker gene expression by qRT-PCR. (A) Nupr1 . (B) Rxrg . (C) Nr4a2 . (D) Pou4f1 . In all assays, expression of the recombinant transcription factor was confirmed by targeted qRT-PCR (data not shown). Gene expression was normalized to an internal Gapdh control, and the fold change over empty vector controls was calculated using the ΔΔ Ct method (see “Materials and Methods”). Gapdh expression levels were stable across all assayed conditions, with no significant changes in Gapdh expression in any of the transfected samples. Error bars indicate 1 SD. n = 5 for all experiments. * P

    Journal: Endocrinology

    Article Title: Transcriptome Analyses of Female Somatotropes and Lactotropes Reveal Novel Regulators of Cell Identity in the Pituitary

    doi: 10.1210/en.2018-00587

    Figure Lengend Snippet: ) were transfected with expression constructs encoding each noted transcription factor linked by an IRES to a GFP reporter open reading frame (see “Materials and Methods”). Transfected cells were isolated by GFP FACS and their RNA content was assayed for specific marker gene expression by qRT-PCR. (A) Nupr1 . (B) Rxrg . (C) Nr4a2 . (D) Pou4f1 . In all assays, expression of the recombinant transcription factor was confirmed by targeted qRT-PCR (data not shown). Gene expression was normalized to an internal Gapdh control, and the fold change over empty vector controls was calculated using the ΔΔ Ct method (see “Materials and Methods”). Gapdh expression levels were stable across all assayed conditions, with no significant changes in Gapdh expression in any of the transfected samples. Error bars indicate 1 SD. n = 5 for all experiments. * P

    Article Snippet: RNA from fluorescence-activated cell sorting (FACS)–sorted cells was isolated (TRIzol LS protocol; Thermo Fisher Scientific) using 10 µg of GlycoBlue coprecipitant (Thermo Fisher Scientific) as a carrier.

    Techniques: Transfection, Expressing, Construct, Isolation, FACS, Marker, Quantitative RT-PCR, Recombinant, Plasmid Preparation

    Characterization of the association between Gag and HIV-1 RNA . (A) Measurement of the association between Gag and HIV-1 genomic RNA. HIV-1 genomic RNA immunoprecipitated with the Gag was characterized by qPCR using a primer/probe set targeting the unspliced RNA transcript. (B) Measurement of the association between Gag and spliced HIV-1 mRNA. The same cDNA preparation described above was subjected to qPCR using a primer/probe set specific for env mRNA and rev mRNA sequences. The copy number in each sample was adjusted for input by the cell number and for transfection efficiency by GFP expression from a co-transfected reporter construct. The amount of NL4-3 RNA was set at 100%. Means and SD of three independent experiments are shown. *, indicates p

    Journal: Retrovirology

    Article Title: Mutations in matrix and SP1 repair the packaging specificity of a Human Immunodeficiency Virus Type 1 mutant by reducing the association of Gag with spliced viral RNA

    doi: 10.1186/1742-4690-7-73

    Figure Lengend Snippet: Characterization of the association between Gag and HIV-1 RNA . (A) Measurement of the association between Gag and HIV-1 genomic RNA. HIV-1 genomic RNA immunoprecipitated with the Gag was characterized by qPCR using a primer/probe set targeting the unspliced RNA transcript. (B) Measurement of the association between Gag and spliced HIV-1 mRNA. The same cDNA preparation described above was subjected to qPCR using a primer/probe set specific for env mRNA and rev mRNA sequences. The copy number in each sample was adjusted for input by the cell number and for transfection efficiency by GFP expression from a co-transfected reporter construct. The amount of NL4-3 RNA was set at 100%. Means and SD of three independent experiments are shown. *, indicates p

    Article Snippet: The resulting viral RNA was converted to cDNA with random hexamers using SuperScript III reverse transcriptase (Invitrogen) and treated with Dpn I.

    Techniques: Immunoprecipitation, Real-time Polymerase Chain Reaction, Transfection, Expressing, Construct