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  • 99
    New England Biolabs polyadenylated
    Polyadenylated, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyadenylated/product/New England Biolabs
    Average 99 stars, based on 65 article reviews
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    89
    Thermo Fisher polyadenylated
    (A) Schematic representation of transcripts, primer sites, siRNA target sites and other genetic elements associated with Oct4 and Oct4-pg5. (B) PCR of MCF-7 RNA depleted of <t>polyadenylated</t> transcripts and converted to cDNA in a strand-specific manner using indicated primers. PCR products were analyzed on 6% polyacrylamide gel. Non-polyadenylated antisense transcripts were detected overlapping the coding region of Oct4-pg5 and the coding and promoter regions of Oct4.
    Polyadenylated, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 163 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 89 stars, based on 163 article reviews
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    89
    Thermo Fisher polyadenylated rnas
    (A) Schematic representation of transcripts, primer sites, siRNA target sites and other genetic elements associated with Oct4 and Oct4-pg5. (B) PCR of MCF-7 RNA depleted of <t>polyadenylated</t> transcripts and converted to cDNA in a strand-specific manner using indicated primers. PCR products were analyzed on 6% polyacrylamide gel. Non-polyadenylated antisense transcripts were detected overlapping the coding region of Oct4-pg5 and the coding and promoter regions of Oct4.
    Polyadenylated Rnas, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 235 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyadenylated rnas/product/Thermo Fisher
    Average 89 stars, based on 235 article reviews
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    mrna  (TaKaRa)
    99
    TaKaRa mrna
    (A) Multiple-tissue Northern blot hybridized to a β3-specific exon B probe. A 7.0-kb <t>mRNA</t> is predominant in the kidneys, liver and lungs, expressed at lower levels in the skeletal muscle, spleen, brain, and heart, and absent from the testes. An additional 4.0-kb mRNA is restricted to skeletal muscle. (B) The same blot hybridized to a Δβ3 probe at high stringency. A 7.0-kb mRNA predominates in the lungs and spleen and is present at low levels in the brain. A 3.0-kb mRNA is present in the same tissues, and a 1.5-kb transcript is evident in the spleen. (C) The same blot hybridized to a β-actin <t>cDNA</t> to show that similar amounts of intact RNA are loaded in each lane. (D) Multiple-tissue Northern blot hybridized to a β1-specific probe. A 6.2-kb mRNA is predominant in the kidneys, liver, brain, and heart, expressed at lower levels in the skeletal muscle, just detectable in the lungs and spleen, and absent from the testes. (E) The same blot hybridized to β2- and α1/α2-specific probes. A 6.2-kb β2 mRNA is clearly expressed in the brain and is just detectable in the lungs and heart but absent from other tissues. The 5.5- and 2.6-kb TRα1 and TRα2 transcripts were expressed at the highest levels in the brain and at lower levels in the kidneys, skeletal muscle, lungs, and heart, were barely detectable in the testes and liver, and were absent from the spleen. (F) Same blot as in panels D and E hybridized to a β-actin cDNA.
    Mrna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 12806 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Illumina Inc polyadenylated
    The balance between ncRNA and mRNA production is a critical aspect of the balance between transcriptional silencing and activation. ( A ) IES-specific <t>polyadenylated</t> transcripts are only produced during developing MAC formation. (3) 3 h after mixing: meiosis, (6) 6 h after mixing: gametogenesis, (10) 10 h after mixing: developing MAC formation, (blue bar) IES. RNA-seq was performed after oligo-dT enrichment of polyadenylated transcripts. Note that essentially no RNA-seq reads were mapped to the IES region at the two early conjugation time points. (PM) Parental MAC, (DM) developing MAC, (OM) old MAC. ( B ) Percentage of RNA-seq reads mapped to consistently processed IESs, relative to total mappable reads. Note that IES-specific polyadenylated transcripts were abundantly produced during developing MAC formation (10 h after mixing) but rarely detected before that (3 and 6 h after mixing). ( C ) Localization of the transcriptional and cotranscriptional machineries in early (3 h after mixing) and late (10 h after mixing) conjugating cells. RPB3-HA, CBP20-HA, PRP19-HA, and THO2-HA cells were stained with an anti-HA antibody (red) and counterstained with DAPI (blue). Parental MAC (PM): green arrowhead, developing MAC (DM): green arrows, MIC: white arrowheads, old MAC (OM): white arrow. ( D ) Interaction between TWI1 and the transcriptional/cotranscriptional machineries. The designated cells were processed for crosslink-immunoprecipitation with the anti-HA antibody at late conjugation (10 h after mixing). The anti-HA and anti-TWI1 antibodies were used for immunoblotting. Note that similar amounts of bait proteins were recovered, as shown by the anti-HA immunoblotting. ( E ) Production of scnRNA from intronic as well as exonic regions. Distributions of polyadenylated transcripts (dotted lines) and scnRNA (solid lines) in introns and exons of IES-specific loci. Each intron is equally divided into 10 units from the 5′ to 3′ end; the two flanking exons are also equally divided into 10 units, respectively. The average RPM of each unit in all the loci were cumulated. Double counting for exons is avoided. ( F ) Alternative production of ncRNA and mRNA in the meiotic MIC and the developing MAC. In this simplified schematic, transcription from IES-specific loci is emphasized, while genic transcription from MDS is omitted. See text for details.
    Polyadenylated, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 91/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyadenylated/product/Illumina Inc
    Average 91 stars, based on 21 article reviews
    Price from $9.99 to $1999.99
    polyadenylated - by Bioz Stars, 2020-08
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    91
    TaKaRa polyadenylated
    The balance between ncRNA and mRNA production is a critical aspect of the balance between transcriptional silencing and activation. ( A ) IES-specific <t>polyadenylated</t> transcripts are only produced during developing MAC formation. (3) 3 h after mixing: meiosis, (6) 6 h after mixing: gametogenesis, (10) 10 h after mixing: developing MAC formation, (blue bar) IES. RNA-seq was performed after oligo-dT enrichment of polyadenylated transcripts. Note that essentially no RNA-seq reads were mapped to the IES region at the two early conjugation time points. (PM) Parental MAC, (DM) developing MAC, (OM) old MAC. ( B ) Percentage of RNA-seq reads mapped to consistently processed IESs, relative to total mappable reads. Note that IES-specific polyadenylated transcripts were abundantly produced during developing MAC formation (10 h after mixing) but rarely detected before that (3 and 6 h after mixing). ( C ) Localization of the transcriptional and cotranscriptional machineries in early (3 h after mixing) and late (10 h after mixing) conjugating cells. RPB3-HA, CBP20-HA, PRP19-HA, and THO2-HA cells were stained with an anti-HA antibody (red) and counterstained with DAPI (blue). Parental MAC (PM): green arrowhead, developing MAC (DM): green arrows, MIC: white arrowheads, old MAC (OM): white arrow. ( D ) Interaction between TWI1 and the transcriptional/cotranscriptional machineries. The designated cells were processed for crosslink-immunoprecipitation with the anti-HA antibody at late conjugation (10 h after mixing). The anti-HA and anti-TWI1 antibodies were used for immunoblotting. Note that similar amounts of bait proteins were recovered, as shown by the anti-HA immunoblotting. ( E ) Production of scnRNA from intronic as well as exonic regions. Distributions of polyadenylated transcripts (dotted lines) and scnRNA (solid lines) in introns and exons of IES-specific loci. Each intron is equally divided into 10 units from the 5′ to 3′ end; the two flanking exons are also equally divided into 10 units, respectively. The average RPM of each unit in all the loci were cumulated. Double counting for exons is avoided. ( F ) Alternative production of ncRNA and mRNA in the meiotic MIC and the developing MAC. In this simplified schematic, transcription from IES-specific loci is emphasized, while genic transcription from MDS is omitted. See text for details.
    Polyadenylated, supplied by TaKaRa, used in various techniques. Bioz Stars score: 91/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyadenylated/product/TaKaRa
    Average 91 stars, based on 39 article reviews
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    polyadenylated - by Bioz Stars, 2020-08
    91/100 stars
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    85
    Agilent technologies viral polyadenylated rnas
    The balance between ncRNA and mRNA production is a critical aspect of the balance between transcriptional silencing and activation. ( A ) IES-specific <t>polyadenylated</t> transcripts are only produced during developing MAC formation. (3) 3 h after mixing: meiosis, (6) 6 h after mixing: gametogenesis, (10) 10 h after mixing: developing MAC formation, (blue bar) IES. RNA-seq was performed after oligo-dT enrichment of polyadenylated transcripts. Note that essentially no RNA-seq reads were mapped to the IES region at the two early conjugation time points. (PM) Parental MAC, (DM) developing MAC, (OM) old MAC. ( B ) Percentage of RNA-seq reads mapped to consistently processed IESs, relative to total mappable reads. Note that IES-specific polyadenylated transcripts were abundantly produced during developing MAC formation (10 h after mixing) but rarely detected before that (3 and 6 h after mixing). ( C ) Localization of the transcriptional and cotranscriptional machineries in early (3 h after mixing) and late (10 h after mixing) conjugating cells. RPB3-HA, CBP20-HA, PRP19-HA, and THO2-HA cells were stained with an anti-HA antibody (red) and counterstained with DAPI (blue). Parental MAC (PM): green arrowhead, developing MAC (DM): green arrows, MIC: white arrowheads, old MAC (OM): white arrow. ( D ) Interaction between TWI1 and the transcriptional/cotranscriptional machineries. The designated cells were processed for crosslink-immunoprecipitation with the anti-HA antibody at late conjugation (10 h after mixing). The anti-HA and anti-TWI1 antibodies were used for immunoblotting. Note that similar amounts of bait proteins were recovered, as shown by the anti-HA immunoblotting. ( E ) Production of scnRNA from intronic as well as exonic regions. Distributions of polyadenylated transcripts (dotted lines) and scnRNA (solid lines) in introns and exons of IES-specific loci. Each intron is equally divided into 10 units from the 5′ to 3′ end; the two flanking exons are also equally divided into 10 units, respectively. The average RPM of each unit in all the loci were cumulated. Double counting for exons is avoided. ( F ) Alternative production of ncRNA and mRNA in the meiotic MIC and the developing MAC. In this simplified schematic, transcription from IES-specific loci is emphasized, while genic transcription from MDS is omitted. See text for details.
    Viral Polyadenylated Rnas, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 85/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/viral polyadenylated rnas/product/Agilent technologies
    Average 85 stars, based on 10 article reviews
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    viral polyadenylated rnas - by Bioz Stars, 2020-08
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    89
    System Biosciences Inc polyadenylated
    The balance between ncRNA and mRNA production is a critical aspect of the balance between transcriptional silencing and activation. ( A ) IES-specific <t>polyadenylated</t> transcripts are only produced during developing MAC formation. (3) 3 h after mixing: meiosis, (6) 6 h after mixing: gametogenesis, (10) 10 h after mixing: developing MAC formation, (blue bar) IES. RNA-seq was performed after oligo-dT enrichment of polyadenylated transcripts. Note that essentially no RNA-seq reads were mapped to the IES region at the two early conjugation time points. (PM) Parental MAC, (DM) developing MAC, (OM) old MAC. ( B ) Percentage of RNA-seq reads mapped to consistently processed IESs, relative to total mappable reads. Note that IES-specific polyadenylated transcripts were abundantly produced during developing MAC formation (10 h after mixing) but rarely detected before that (3 and 6 h after mixing). ( C ) Localization of the transcriptional and cotranscriptional machineries in early (3 h after mixing) and late (10 h after mixing) conjugating cells. RPB3-HA, CBP20-HA, PRP19-HA, and THO2-HA cells were stained with an anti-HA antibody (red) and counterstained with DAPI (blue). Parental MAC (PM): green arrowhead, developing MAC (DM): green arrows, MIC: white arrowheads, old MAC (OM): white arrow. ( D ) Interaction between TWI1 and the transcriptional/cotranscriptional machineries. The designated cells were processed for crosslink-immunoprecipitation with the anti-HA antibody at late conjugation (10 h after mixing). The anti-HA and anti-TWI1 antibodies were used for immunoblotting. Note that similar amounts of bait proteins were recovered, as shown by the anti-HA immunoblotting. ( E ) Production of scnRNA from intronic as well as exonic regions. Distributions of polyadenylated transcripts (dotted lines) and scnRNA (solid lines) in introns and exons of IES-specific loci. Each intron is equally divided into 10 units from the 5′ to 3′ end; the two flanking exons are also equally divided into 10 units, respectively. The average RPM of each unit in all the loci were cumulated. Double counting for exons is avoided. ( F ) Alternative production of ncRNA and mRNA in the meiotic MIC and the developing MAC. In this simplified schematic, transcription from IES-specific loci is emphasized, while genic transcription from MDS is omitted. See text for details.
    Polyadenylated, supplied by System Biosciences Inc, used in various techniques. Bioz Stars score: 89/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyadenylated/product/System Biosciences Inc
    Average 89 stars, based on 4 article reviews
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    polyadenylated - by Bioz Stars, 2020-08
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    96
    Thermo Fisher mrna
    Transfection of non-phagocytic cells, using <t>mRNA:LPNs</t> and mRNA:CS-PLGA at different ratios performed in epithelial A549 cells for 24 h and 48 h post-transfection using flow cytometer. mRNA complexed LPNs reveal a significant higher transgene expression over CS-PLGA NPs. Both NPs show higher transfection rates with higher mRNA:NP ratios and increasing transfection until 48 h post-transfection N = 4, mean ± SD (*p
    Mrna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 89710 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mrna/product/Thermo Fisher
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    92
    TriLink polyadenylated cas9 mrna
    Transfection of non-phagocytic cells, using <t>mRNA:LPNs</t> and mRNA:CS-PLGA at different ratios performed in epithelial A549 cells for 24 h and 48 h post-transfection using flow cytometer. mRNA complexed LPNs reveal a significant higher transgene expression over CS-PLGA NPs. Both NPs show higher transfection rates with higher mRNA:NP ratios and increasing transfection until 48 h post-transfection N = 4, mean ± SD (*p
    Polyadenylated Cas9 Mrna, supplied by TriLink, used in various techniques. Bioz Stars score: 92/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Illumina Inc polyadenylated rnas
    Location of cto genes on the PRV genome. Both cto transcripts (CTOs) are <t>polyadenylated</t> <t>RNAs</t> with a common 3′ termination. CTO-L is generated by the continuation of transcription after the termination signals of the ul21 gene. OriL is the replication origin of in the UL region of viral DNA mapped between the ul21 and ul22 genes.
    Polyadenylated Rnas, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 91/100, based on 76 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyadenylated rnas/product/Illumina Inc
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    99
    New England Biolabs mrna
    Transcriptome profiling to explore the mechanism of the synthetic lethality. (A) and (B) REACTOME signal pathway analysis for the <t>RNA</t> sequencing results is shown. HCC1937 BRCA1 isogenic cells were treated with 5 μmol/L entinostat for 24 h and total RNA was extracted for RNA sequencing. The gene expression profiles were analyzed with REACTOME signal pathway database. The pathway genes that were significantly up- (A) and down-regulated (B) by entinostat are shown. (C) and (D) The effect of entinostat on cellular oxidative stress and DNA damage was examined. HCC1937 BRCA1 −/− (C) or T47D sh BRCA1 (D) cells were treated with 5 μmol/L entinostat for indicated time points and Western blots for proteins involved in oxidative stress and DNA damage responses were analyzed. GAPDH was used as an internal control. (E) RT-qPCR analysis for the TXNIP <t>mRNA</t> level was examined in HCC1937 BRCA1 isogenic cells treated with entinostat. (F) Chromatin immunoprecipitation (ChIP) of TXNIP promoter using anti-acetyl histone H4 antibody is shown. HCC1937 BRCA1 −/− cells were treated with 5 μmol/L entinostat for 6 h and processed for ChIP using a rabbit IgG (control) or anti-acetyl histone H4 antibody. The ChIP DNA was subjected to PCR amplification with a primer pair specific for TXNIP promoter. (G) and (I) The effect of HDAC inhibitors, entinostat (class I-HDACi, G), vorinostat (pan-HDACi, H), and mocetinostat (class I-HDACi, I) on histone acetylation, cellular oxidative stress and DNA damage responses was examined in HCC1937 BRCA1 isogenic cell lines. Data are mean ± SD, * P
    Mrna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 7791 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mrna/product/New England Biolabs
    Average 99 stars, based on 7791 article reviews
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    mrna - by Bioz Stars, 2020-08
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    89
    GE Healthcare polyadenylated transcripts
    The balance between ncRNA and mRNA production is a critical aspect of the balance between transcriptional silencing and activation. ( A ) IES-specific <t>polyadenylated</t> transcripts are only produced during developing MAC formation. (3) 3 h after mixing: meiosis, (6) 6 h after mixing: gametogenesis, (10) 10 h after mixing: developing MAC formation, (blue bar) IES. RNA-seq was performed after oligo-dT enrichment of polyadenylated transcripts. Note that essentially no RNA-seq reads were mapped to the IES region at the two early conjugation time points. (PM) Parental MAC, (DM) developing MAC, (OM) old MAC. ( B ) Percentage of RNA-seq reads mapped to consistently processed IESs, relative to total mappable reads. Note that IES-specific polyadenylated transcripts were abundantly produced during developing MAC formation (10 h after mixing) but rarely detected before that (3 and 6 h after mixing). ( C ) Localization of the transcriptional and cotranscriptional machineries in early (3 h after mixing) and late (10 h after mixing) conjugating cells. RPB3-HA, CBP20-HA, PRP19-HA, and THO2-HA cells were stained with an anti-HA antibody (red) and counterstained with DAPI (blue). Parental MAC (PM): green arrowhead, developing MAC (DM): green arrows, MIC: white arrowheads, old MAC (OM): white arrow. ( D ) Interaction between TWI1 and the transcriptional/cotranscriptional machineries. The designated cells were processed for crosslink-immunoprecipitation with the anti-HA antibody at late conjugation (10 h after mixing). The anti-HA and anti-TWI1 antibodies were used for immunoblotting. Note that similar amounts of bait proteins were recovered, as shown by the anti-HA immunoblotting. ( E ) Production of scnRNA from intronic as well as exonic regions. Distributions of polyadenylated transcripts (dotted lines) and scnRNA (solid lines) in introns and exons of IES-specific loci. Each intron is equally divided into 10 units from the 5′ to 3′ end; the two flanking exons are also equally divided into 10 units, respectively. The average RPM of each unit in all the loci were cumulated. Double counting for exons is avoided. ( F ) Alternative production of ncRNA and mRNA in the meiotic MIC and the developing MAC. In this simplified schematic, transcription from IES-specific loci is emphasized, while genic transcription from MDS is omitted. See text for details.
    Polyadenylated Transcripts, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 89/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyadenylated transcripts/product/GE Healthcare
    Average 89 stars, based on 14 article reviews
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    polyadenylated transcripts - by Bioz Stars, 2020-08
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    89
    Agilent technologies polyadenylated transcripts
    The balance between ncRNA and mRNA production is a critical aspect of the balance between transcriptional silencing and activation. ( A ) IES-specific <t>polyadenylated</t> transcripts are only produced during developing MAC formation. (3) 3 h after mixing: meiosis, (6) 6 h after mixing: gametogenesis, (10) 10 h after mixing: developing MAC formation, (blue bar) IES. RNA-seq was performed after oligo-dT enrichment of polyadenylated transcripts. Note that essentially no RNA-seq reads were mapped to the IES region at the two early conjugation time points. (PM) Parental MAC, (DM) developing MAC, (OM) old MAC. ( B ) Percentage of RNA-seq reads mapped to consistently processed IESs, relative to total mappable reads. Note that IES-specific polyadenylated transcripts were abundantly produced during developing MAC formation (10 h after mixing) but rarely detected before that (3 and 6 h after mixing). ( C ) Localization of the transcriptional and cotranscriptional machineries in early (3 h after mixing) and late (10 h after mixing) conjugating cells. RPB3-HA, CBP20-HA, PRP19-HA, and THO2-HA cells were stained with an anti-HA antibody (red) and counterstained with DAPI (blue). Parental MAC (PM): green arrowhead, developing MAC (DM): green arrows, MIC: white arrowheads, old MAC (OM): white arrow. ( D ) Interaction between TWI1 and the transcriptional/cotranscriptional machineries. The designated cells were processed for crosslink-immunoprecipitation with the anti-HA antibody at late conjugation (10 h after mixing). The anti-HA and anti-TWI1 antibodies were used for immunoblotting. Note that similar amounts of bait proteins were recovered, as shown by the anti-HA immunoblotting. ( E ) Production of scnRNA from intronic as well as exonic regions. Distributions of polyadenylated transcripts (dotted lines) and scnRNA (solid lines) in introns and exons of IES-specific loci. Each intron is equally divided into 10 units from the 5′ to 3′ end; the two flanking exons are also equally divided into 10 units, respectively. The average RPM of each unit in all the loci were cumulated. Double counting for exons is avoided. ( F ) Alternative production of ncRNA and mRNA in the meiotic MIC and the developing MAC. In this simplified schematic, transcription from IES-specific loci is emphasized, while genic transcription from MDS is omitted. See text for details.
    Polyadenylated Transcripts, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 89/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyadenylated transcripts/product/Agilent technologies
    Average 89 stars, based on 11 article reviews
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    polyadenylated transcripts - by Bioz Stars, 2020-08
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    90
    Lonza polyadenylated mrnas
    The balance between ncRNA and mRNA production is a critical aspect of the balance between transcriptional silencing and activation. ( A ) IES-specific <t>polyadenylated</t> transcripts are only produced during developing MAC formation. (3) 3 h after mixing: meiosis, (6) 6 h after mixing: gametogenesis, (10) 10 h after mixing: developing MAC formation, (blue bar) IES. RNA-seq was performed after oligo-dT enrichment of polyadenylated transcripts. Note that essentially no RNA-seq reads were mapped to the IES region at the two early conjugation time points. (PM) Parental MAC, (DM) developing MAC, (OM) old MAC. ( B ) Percentage of RNA-seq reads mapped to consistently processed IESs, relative to total mappable reads. Note that IES-specific polyadenylated transcripts were abundantly produced during developing MAC formation (10 h after mixing) but rarely detected before that (3 and 6 h after mixing). ( C ) Localization of the transcriptional and cotranscriptional machineries in early (3 h after mixing) and late (10 h after mixing) conjugating cells. RPB3-HA, CBP20-HA, PRP19-HA, and THO2-HA cells were stained with an anti-HA antibody (red) and counterstained with DAPI (blue). Parental MAC (PM): green arrowhead, developing MAC (DM): green arrows, MIC: white arrowheads, old MAC (OM): white arrow. ( D ) Interaction between TWI1 and the transcriptional/cotranscriptional machineries. The designated cells were processed for crosslink-immunoprecipitation with the anti-HA antibody at late conjugation (10 h after mixing). The anti-HA and anti-TWI1 antibodies were used for immunoblotting. Note that similar amounts of bait proteins were recovered, as shown by the anti-HA immunoblotting. ( E ) Production of scnRNA from intronic as well as exonic regions. Distributions of polyadenylated transcripts (dotted lines) and scnRNA (solid lines) in introns and exons of IES-specific loci. Each intron is equally divided into 10 units from the 5′ to 3′ end; the two flanking exons are also equally divided into 10 units, respectively. The average RPM of each unit in all the loci were cumulated. Double counting for exons is avoided. ( F ) Alternative production of ncRNA and mRNA in the meiotic MIC and the developing MAC. In this simplified schematic, transcription from IES-specific loci is emphasized, while genic transcription from MDS is omitted. See text for details.
    Polyadenylated Mrnas, supplied by Lonza, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyadenylated mrnas/product/Lonza
    Average 90 stars, based on 7 article reviews
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    polyadenylated mrnas - by Bioz Stars, 2020-08
    90/100 stars
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    85
    Agilent technologies synthetic polyadenylated transcripts
    The balance between ncRNA and mRNA production is a critical aspect of the balance between transcriptional silencing and activation. ( A ) IES-specific <t>polyadenylated</t> transcripts are only produced during developing MAC formation. (3) 3 h after mixing: meiosis, (6) 6 h after mixing: gametogenesis, (10) 10 h after mixing: developing MAC formation, (blue bar) IES. RNA-seq was performed after oligo-dT enrichment of polyadenylated transcripts. Note that essentially no RNA-seq reads were mapped to the IES region at the two early conjugation time points. (PM) Parental MAC, (DM) developing MAC, (OM) old MAC. ( B ) Percentage of RNA-seq reads mapped to consistently processed IESs, relative to total mappable reads. Note that IES-specific polyadenylated transcripts were abundantly produced during developing MAC formation (10 h after mixing) but rarely detected before that (3 and 6 h after mixing). ( C ) Localization of the transcriptional and cotranscriptional machineries in early (3 h after mixing) and late (10 h after mixing) conjugating cells. RPB3-HA, CBP20-HA, PRP19-HA, and THO2-HA cells were stained with an anti-HA antibody (red) and counterstained with DAPI (blue). Parental MAC (PM): green arrowhead, developing MAC (DM): green arrows, MIC: white arrowheads, old MAC (OM): white arrow. ( D ) Interaction between TWI1 and the transcriptional/cotranscriptional machineries. The designated cells were processed for crosslink-immunoprecipitation with the anti-HA antibody at late conjugation (10 h after mixing). The anti-HA and anti-TWI1 antibodies were used for immunoblotting. Note that similar amounts of bait proteins were recovered, as shown by the anti-HA immunoblotting. ( E ) Production of scnRNA from intronic as well as exonic regions. Distributions of polyadenylated transcripts (dotted lines) and scnRNA (solid lines) in introns and exons of IES-specific loci. Each intron is equally divided into 10 units from the 5′ to 3′ end; the two flanking exons are also equally divided into 10 units, respectively. The average RPM of each unit in all the loci were cumulated. Double counting for exons is avoided. ( F ) Alternative production of ncRNA and mRNA in the meiotic MIC and the developing MAC. In this simplified schematic, transcription from IES-specific loci is emphasized, while genic transcription from MDS is omitted. See text for details.
    Synthetic Polyadenylated Transcripts, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/synthetic polyadenylated transcripts/product/Agilent technologies
    Average 85 stars, based on 2 article reviews
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    synthetic polyadenylated transcripts - by Bioz Stars, 2020-08
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    85
    Illumina Inc polyadenylated 3
    The balance between ncRNA and mRNA production is a critical aspect of the balance between transcriptional silencing and activation. ( A ) IES-specific <t>polyadenylated</t> transcripts are only produced during developing MAC formation. (3) 3 h after mixing: meiosis, (6) 6 h after mixing: gametogenesis, (10) 10 h after mixing: developing MAC formation, (blue bar) IES. RNA-seq was performed after oligo-dT enrichment of polyadenylated transcripts. Note that essentially no RNA-seq reads were mapped to the IES region at the two early conjugation time points. (PM) Parental MAC, (DM) developing MAC, (OM) old MAC. ( B ) Percentage of RNA-seq reads mapped to consistently processed IESs, relative to total mappable reads. Note that IES-specific polyadenylated transcripts were abundantly produced during developing MAC formation (10 h after mixing) but rarely detected before that (3 and 6 h after mixing). ( C ) Localization of the transcriptional and cotranscriptional machineries in early (3 h after mixing) and late (10 h after mixing) conjugating cells. RPB3-HA, CBP20-HA, PRP19-HA, and THO2-HA cells were stained with an anti-HA antibody (red) and counterstained with DAPI (blue). Parental MAC (PM): green arrowhead, developing MAC (DM): green arrows, MIC: white arrowheads, old MAC (OM): white arrow. ( D ) Interaction between TWI1 and the transcriptional/cotranscriptional machineries. The designated cells were processed for crosslink-immunoprecipitation with the anti-HA antibody at late conjugation (10 h after mixing). The anti-HA and anti-TWI1 antibodies were used for immunoblotting. Note that similar amounts of bait proteins were recovered, as shown by the anti-HA immunoblotting. ( E ) Production of scnRNA from intronic as well as exonic regions. Distributions of polyadenylated transcripts (dotted lines) and scnRNA (solid lines) in introns and exons of IES-specific loci. Each intron is equally divided into 10 units from the 5′ to 3′ end; the two flanking exons are also equally divided into 10 units, respectively. The average RPM of each unit in all the loci were cumulated. Double counting for exons is avoided. ( F ) Alternative production of ncRNA and mRNA in the meiotic MIC and the developing MAC. In this simplified schematic, transcription from IES-specific loci is emphasized, while genic transcription from MDS is omitted. See text for details.
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    GE Healthcare mrna
    Stimulation of <t>HIEC/IEC-6</t> cells with LPS induced TNFα, as demonstrated by RT-PCR (A). Already after a stimulation period of three hours TNFα <t>mRNA</t> transcripts were observed in both IEC models. The expression of G3PDH was used as house keeping gene. The translation into the protein product and subsequent secretion of TNFα into the culture medium was analysed in HIEC using a high sensitivity ELISA (B). TNFα was produced in a dose dependent manner after stimulation with LPS. CHX treatment completely suppressed the secretion of TNFα. In keeping, functional experiments with CHX showed in HIEC that the proliferation rate after LPS stimulation remained unchanged once TNFα production was suppressed (C). On the other hand, addition of TNFα in the presence of CHX was still able to suppress HIEC proliferation. Concentrations (x axis) for CHX or LPS in μg/ml, for TNFα in ng/ml. The concentrations of CHX used together with LPS or TNFα were 1 μg/ml.
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    Illumina Inc mrna
    microHIVE differentiation of spinal motor neurons. (A) Target and experimental molecular profiles of retinoic acid and GDF11. (Left) Based on correlative analysis of growth factor concentrations and <t>RNA</t> profiling, we designed a continuous profile of retinoic acid and GDF11 to induce rostral-caudal patterning of motor neurons, to correspond to the brachial, thoracic and lumbar regions of the spinal cord. (Right) The microHIVE experimental profile showed a close correlation to the desired target profile (profile similarity metric, ε = 0.991). (B) Schematic illustration of chamber binning. After motor neuron differentiation (day 28), the all-polymer cell culture chamber could be sectioned into seven bins for cellular characterization and spatial correlation. (C) Differential expressions of HOX genes across the chamber bins. HOX gene expressions were profiled from the respective chamber sections through quantitative PCR. All gene expression analyses were made relative to intrinsic GAPDH <t>mRNA</t> levels, and subsequently gene (column) normalized across all bins to compare respective gene expression trends in the form of a heat map. (D) Immunofluorescence confirmation of HOX protein expressions. Immunostaining of HOXB4 and HOXC8 (red) and SMI-32 (green) across the seven culture bins demonstrated successful generation of rostral (HOXB4) to caudal (HOXC8) motor neurons. Cellular nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. Cellular quantification based on positive staining of HOXB4 and HOXC8 further confirmed the respective protein expression trends. All measurements were performed in triplicate, and the data are displayed as mean ± s.d. in A and D .
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    Image Search Results


    (A) Schematic representation of transcripts, primer sites, siRNA target sites and other genetic elements associated with Oct4 and Oct4-pg5. (B) PCR of MCF-7 RNA depleted of polyadenylated transcripts and converted to cDNA in a strand-specific manner using indicated primers. PCR products were analyzed on 6% polyacrylamide gel. Non-polyadenylated antisense transcripts were detected overlapping the coding region of Oct4-pg5 and the coding and promoter regions of Oct4.

    Journal: Transcription

    Article Title: Transcriptional regulation of Oct4 by a long non-coding RNA antisense to Oct4-pseudogene 5

    doi: 10.4161/trns.1.3.13332

    Figure Lengend Snippet: (A) Schematic representation of transcripts, primer sites, siRNA target sites and other genetic elements associated with Oct4 and Oct4-pg5. (B) PCR of MCF-7 RNA depleted of polyadenylated transcripts and converted to cDNA in a strand-specific manner using indicated primers. PCR products were analyzed on 6% polyacrylamide gel. Non-polyadenylated antisense transcripts were detected overlapping the coding region of Oct4-pg5 and the coding and promoter regions of Oct4.

    Article Snippet: MCF-7 RNA was isolated (RNeasy, Qaigen™), DNase treated (TURBO DNase, Ambion™), depleted of polyadenylated transcripts (mRNA Catcher, Invitrogen™) and converted to cDNA (Reverse Transcrpition Core Kit, Eurogentec™) using indicated primers. cDNA was amplified by PCR (KAPA2G Fast HotStart, Kapa Biosystems) using indicated primers and analyzed on a 6% polyacrylamide gel.

    Techniques: Polymerase Chain Reaction

    (A) Multiple-tissue Northern blot hybridized to a β3-specific exon B probe. A 7.0-kb mRNA is predominant in the kidneys, liver and lungs, expressed at lower levels in the skeletal muscle, spleen, brain, and heart, and absent from the testes. An additional 4.0-kb mRNA is restricted to skeletal muscle. (B) The same blot hybridized to a Δβ3 probe at high stringency. A 7.0-kb mRNA predominates in the lungs and spleen and is present at low levels in the brain. A 3.0-kb mRNA is present in the same tissues, and a 1.5-kb transcript is evident in the spleen. (C) The same blot hybridized to a β-actin cDNA to show that similar amounts of intact RNA are loaded in each lane. (D) Multiple-tissue Northern blot hybridized to a β1-specific probe. A 6.2-kb mRNA is predominant in the kidneys, liver, brain, and heart, expressed at lower levels in the skeletal muscle, just detectable in the lungs and spleen, and absent from the testes. (E) The same blot hybridized to β2- and α1/α2-specific probes. A 6.2-kb β2 mRNA is clearly expressed in the brain and is just detectable in the lungs and heart but absent from other tissues. The 5.5- and 2.6-kb TRα1 and TRα2 transcripts were expressed at the highest levels in the brain and at lower levels in the kidneys, skeletal muscle, lungs, and heart, were barely detectable in the testes and liver, and were absent from the spleen. (F) Same blot as in panels D and E hybridized to a β-actin cDNA.

    Journal: Molecular and Cellular Biology

    Article Title: Cloning and Characterization of Two Novel Thyroid Hormone Receptor ? Isoforms

    doi:

    Figure Lengend Snippet: (A) Multiple-tissue Northern blot hybridized to a β3-specific exon B probe. A 7.0-kb mRNA is predominant in the kidneys, liver and lungs, expressed at lower levels in the skeletal muscle, spleen, brain, and heart, and absent from the testes. An additional 4.0-kb mRNA is restricted to skeletal muscle. (B) The same blot hybridized to a Δβ3 probe at high stringency. A 7.0-kb mRNA predominates in the lungs and spleen and is present at low levels in the brain. A 3.0-kb mRNA is present in the same tissues, and a 1.5-kb transcript is evident in the spleen. (C) The same blot hybridized to a β-actin cDNA to show that similar amounts of intact RNA are loaded in each lane. (D) Multiple-tissue Northern blot hybridized to a β1-specific probe. A 6.2-kb mRNA is predominant in the kidneys, liver, brain, and heart, expressed at lower levels in the skeletal muscle, just detectable in the lungs and spleen, and absent from the testes. (E) The same blot hybridized to β2- and α1/α2-specific probes. A 6.2-kb β2 mRNA is clearly expressed in the brain and is just detectable in the lungs and heart but absent from other tissues. The 5.5- and 2.6-kb TRα1 and TRα2 transcripts were expressed at the highest levels in the brain and at lower levels in the kidneys, skeletal muscle, lungs, and heart, were barely detectable in the testes and liver, and were absent from the spleen. (F) Same blot as in panels D and E hybridized to a β-actin cDNA.

    Article Snippet: UMR106 and GH3 adapter-ligated cDNA libraries were constructed from poly(A)+ mRNA (Marathon cDNA synthesis; Clontech), and 5′-RACE was performed to identify TRβ variants using primers BR2 and BR1 and Marathon adapter primers, AP1 and AP2.

    Techniques: Northern Blot

    BOULE expression is distinct from that of DAZ and DAZL. DAZ is expressed in prenatal primordial germ cells, spermatogonial stem cells, and spermatocytes. DAZL is expressed in both the male and female germ line. BOULE is expressed in the cytoplasm of pachytene spermatocytes, persists through meiosis, and decreases in early spermatids. ( A ). ( B ). ( C ) Human testis section stained using antisera that recognize DAZ only. DAZ is expressed in spermatogonia and early spermatocytes, but is absent from late spermatocytes or postmeiotic cells. ( D ). ( E ) A Northern blot with polyadenylated RNA from different human tissues. Blot was hybridized with human BOULE cDNA that detects two testes specific transcripts. Lanes: 1, spleen; 2, thymus; 3, prostate; 4, testis; 5, ovary; 6, small intestine; 7, colon; and 8, leukocyte. ( F ) A Northern blot with polyadenylated RNA from mouse tissues. Blot was hybridized with human BOULE cDNA that detects three testes-specific transcripts. Lanes: 1, heart; 2, brain; 3, spleen; 4, lung; 5, liver; 6, muscle; 7, kidney; and 8, testis. ( G ) Anti-BOULE antisera detects a single 32-kDa protein in mouse testes ( b ) and a similar size protein in human testes ( a ) but not in other human or mouse tissues (data not shown), nor does it recognize DAZ protein expressed in yeast strain, RRY618 ( c ). The 50-kDa band in human testes is nonspecific as it is detected by preimmune also. ( H ) BOULE staining in human testis section is also restricted to cytoplasm of spermatocytes; no staining of spermatogonial stem cells is observed. ( I ) Stage III seminiferous tubules. BOULE is expressed in round spermatids (Spd) and secondary spermatocytes but not in spermatogonia (Spg) or primary spermatocytes (Spc). ( J ) Stage VII seminiferous tubule. BOULE is expressed in the cytoplasm of pachytene spermatocytes. There is no staining in spermatogonia and spermatids. ( K ) Stage X–XI seminiferous tubules. BOULE expression peaks in late pachytene stage spermatocytes. ( L and M ) Lower-magnification view of staining with preimmune and anti-BOULE antisera. ( L ) Preimmune of BOULE antisera (×200). ( M ).

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

    Article Title: A gene family required for human germ cell development evolved from an ancient meiotic gene conserved in metazoans

    doi: 10.1073/pnas.131090498

    Figure Lengend Snippet: BOULE expression is distinct from that of DAZ and DAZL. DAZ is expressed in prenatal primordial germ cells, spermatogonial stem cells, and spermatocytes. DAZL is expressed in both the male and female germ line. BOULE is expressed in the cytoplasm of pachytene spermatocytes, persists through meiosis, and decreases in early spermatids. ( A ). ( B ). ( C ) Human testis section stained using antisera that recognize DAZ only. DAZ is expressed in spermatogonia and early spermatocytes, but is absent from late spermatocytes or postmeiotic cells. ( D ). ( E ) A Northern blot with polyadenylated RNA from different human tissues. Blot was hybridized with human BOULE cDNA that detects two testes specific transcripts. Lanes: 1, spleen; 2, thymus; 3, prostate; 4, testis; 5, ovary; 6, small intestine; 7, colon; and 8, leukocyte. ( F ) A Northern blot with polyadenylated RNA from mouse tissues. Blot was hybridized with human BOULE cDNA that detects three testes-specific transcripts. Lanes: 1, heart; 2, brain; 3, spleen; 4, lung; 5, liver; 6, muscle; 7, kidney; and 8, testis. ( G ) Anti-BOULE antisera detects a single 32-kDa protein in mouse testes ( b ) and a similar size protein in human testes ( a ) but not in other human or mouse tissues (data not shown), nor does it recognize DAZ protein expressed in yeast strain, RRY618 ( c ). The 50-kDa band in human testes is nonspecific as it is detected by preimmune also. ( H ) BOULE staining in human testis section is also restricted to cytoplasm of spermatocytes; no staining of spermatogonial stem cells is observed. ( I ) Stage III seminiferous tubules. BOULE is expressed in round spermatids (Spd) and secondary spermatocytes but not in spermatogonia (Spg) or primary spermatocytes (Spc). ( J ) Stage VII seminiferous tubule. BOULE is expressed in the cytoplasm of pachytene spermatocytes. There is no staining in spermatogonia and spermatids. ( K ) Stage X–XI seminiferous tubules. BOULE expression peaks in late pachytene stage spermatocytes. ( L and M ) Lower-magnification view of staining with preimmune and anti-BOULE antisera. ( L ) Preimmune of BOULE antisera (×200). ( M ).

    Article Snippet: Northern hybridization was done on polyadenylated RNA blots (CLONTECH) using human and mouse cDNA clones.

    Techniques: Expressing, Staining, Northern Blot

    The balance between ncRNA and mRNA production is a critical aspect of the balance between transcriptional silencing and activation. ( A ) IES-specific polyadenylated transcripts are only produced during developing MAC formation. (3) 3 h after mixing: meiosis, (6) 6 h after mixing: gametogenesis, (10) 10 h after mixing: developing MAC formation, (blue bar) IES. RNA-seq was performed after oligo-dT enrichment of polyadenylated transcripts. Note that essentially no RNA-seq reads were mapped to the IES region at the two early conjugation time points. (PM) Parental MAC, (DM) developing MAC, (OM) old MAC. ( B ) Percentage of RNA-seq reads mapped to consistently processed IESs, relative to total mappable reads. Note that IES-specific polyadenylated transcripts were abundantly produced during developing MAC formation (10 h after mixing) but rarely detected before that (3 and 6 h after mixing). ( C ) Localization of the transcriptional and cotranscriptional machineries in early (3 h after mixing) and late (10 h after mixing) conjugating cells. RPB3-HA, CBP20-HA, PRP19-HA, and THO2-HA cells were stained with an anti-HA antibody (red) and counterstained with DAPI (blue). Parental MAC (PM): green arrowhead, developing MAC (DM): green arrows, MIC: white arrowheads, old MAC (OM): white arrow. ( D ) Interaction between TWI1 and the transcriptional/cotranscriptional machineries. The designated cells were processed for crosslink-immunoprecipitation with the anti-HA antibody at late conjugation (10 h after mixing). The anti-HA and anti-TWI1 antibodies were used for immunoblotting. Note that similar amounts of bait proteins were recovered, as shown by the anti-HA immunoblotting. ( E ) Production of scnRNA from intronic as well as exonic regions. Distributions of polyadenylated transcripts (dotted lines) and scnRNA (solid lines) in introns and exons of IES-specific loci. Each intron is equally divided into 10 units from the 5′ to 3′ end; the two flanking exons are also equally divided into 10 units, respectively. The average RPM of each unit in all the loci were cumulated. Double counting for exons is avoided. ( F ) Alternative production of ncRNA and mRNA in the meiotic MIC and the developing MAC. In this simplified schematic, transcription from IES-specific loci is emphasized, while genic transcription from MDS is omitted. See text for details.

    Journal: Genes & Development

    Article Title: RNAi-dependent Polycomb repression controls transposable elements in Tetrahymena

    doi: 10.1101/gad.320796.118

    Figure Lengend Snippet: The balance between ncRNA and mRNA production is a critical aspect of the balance between transcriptional silencing and activation. ( A ) IES-specific polyadenylated transcripts are only produced during developing MAC formation. (3) 3 h after mixing: meiosis, (6) 6 h after mixing: gametogenesis, (10) 10 h after mixing: developing MAC formation, (blue bar) IES. RNA-seq was performed after oligo-dT enrichment of polyadenylated transcripts. Note that essentially no RNA-seq reads were mapped to the IES region at the two early conjugation time points. (PM) Parental MAC, (DM) developing MAC, (OM) old MAC. ( B ) Percentage of RNA-seq reads mapped to consistently processed IESs, relative to total mappable reads. Note that IES-specific polyadenylated transcripts were abundantly produced during developing MAC formation (10 h after mixing) but rarely detected before that (3 and 6 h after mixing). ( C ) Localization of the transcriptional and cotranscriptional machineries in early (3 h after mixing) and late (10 h after mixing) conjugating cells. RPB3-HA, CBP20-HA, PRP19-HA, and THO2-HA cells were stained with an anti-HA antibody (red) and counterstained with DAPI (blue). Parental MAC (PM): green arrowhead, developing MAC (DM): green arrows, MIC: white arrowheads, old MAC (OM): white arrow. ( D ) Interaction between TWI1 and the transcriptional/cotranscriptional machineries. The designated cells were processed for crosslink-immunoprecipitation with the anti-HA antibody at late conjugation (10 h after mixing). The anti-HA and anti-TWI1 antibodies were used for immunoblotting. Note that similar amounts of bait proteins were recovered, as shown by the anti-HA immunoblotting. ( E ) Production of scnRNA from intronic as well as exonic regions. Distributions of polyadenylated transcripts (dotted lines) and scnRNA (solid lines) in introns and exons of IES-specific loci. Each intron is equally divided into 10 units from the 5′ to 3′ end; the two flanking exons are also equally divided into 10 units, respectively. The average RPM of each unit in all the loci were cumulated. Double counting for exons is avoided. ( F ) Alternative production of ncRNA and mRNA in the meiotic MIC and the developing MAC. In this simplified schematic, transcription from IES-specific loci is emphasized, while genic transcription from MDS is omitted. See text for details.

    Article Snippet: The RNA samples, after oligo-dT enrichment of polyadenylated transcripts, were analyzed by strand-specific Illumina sequencing (RNA sequencing [RNA-seq]).

    Techniques: Activation Assay, Produced, RNA Sequencing Assay, Conjugation Assay, Staining, Immunoprecipitation

    Widespread production of IES-specific polyadenylated transcripts in mutants deficient in RNAi-dependent Polycomb repression. ( A ) A schematic for DNA elimination in Tetrahymena . ( B ) Key steps and players in the DNA elimination pathway. ( C ) from highest to lowest by their coverage of polyadenylated RNA (reads per kilobase of transcript per million mapped reads [RPKM]), in wild type and mutants deficient in RNAi-dependent Polycomb repression ( ΔDCL1, ΔEZL1 , and ΔPDD1 , respectively). ( Inset ) The top 100 IESs. Note increased and widespread transcription of IES-specific sequences in the mutants. ( D ) Distribution of highly expressed IESs, in wild type and the mutants. Supercontig 2.1, over 3.5 Mb in length, is analyzed. Read densities (RPKM) are represented by a color scale. Note the alignment of red stripes in the mutants, indicating coregulation by the RNAi-dependent Polycomb repression pathway. ( E ) A representative GBrowse view illustrating RNA sequencing (RNA-seq) coverage in wild type and the mutants. (Blue bar) IES. Gene model: an IES-specific ( left ) and an MDS-specific ( right ) gene. Note dramatically increased IES transcription in the mutants, compared with wild-type cells. ( F ) A proportional Venn diagram representing IESs highly induced in the mutants. ( G ) A box plot comparing PDD1 enrichment in wild-type cells [(ChIP − input)/(ChIP + input)] in IESs induced in the mutants ( ΔDCL1 , ΔEZL1 , and ΔPDD1 cells, respectively) with IESs not induced in any mutants. The first quartile, median, and the third quartile are marked. A Kruskal-Wallis H test was performed for all three pairwise comparisons, revealing highly significant variances. P

    Journal: Genes & Development

    Article Title: RNAi-dependent Polycomb repression controls transposable elements in Tetrahymena

    doi: 10.1101/gad.320796.118

    Figure Lengend Snippet: Widespread production of IES-specific polyadenylated transcripts in mutants deficient in RNAi-dependent Polycomb repression. ( A ) A schematic for DNA elimination in Tetrahymena . ( B ) Key steps and players in the DNA elimination pathway. ( C ) from highest to lowest by their coverage of polyadenylated RNA (reads per kilobase of transcript per million mapped reads [RPKM]), in wild type and mutants deficient in RNAi-dependent Polycomb repression ( ΔDCL1, ΔEZL1 , and ΔPDD1 , respectively). ( Inset ) The top 100 IESs. Note increased and widespread transcription of IES-specific sequences in the mutants. ( D ) Distribution of highly expressed IESs, in wild type and the mutants. Supercontig 2.1, over 3.5 Mb in length, is analyzed. Read densities (RPKM) are represented by a color scale. Note the alignment of red stripes in the mutants, indicating coregulation by the RNAi-dependent Polycomb repression pathway. ( E ) A representative GBrowse view illustrating RNA sequencing (RNA-seq) coverage in wild type and the mutants. (Blue bar) IES. Gene model: an IES-specific ( left ) and an MDS-specific ( right ) gene. Note dramatically increased IES transcription in the mutants, compared with wild-type cells. ( F ) A proportional Venn diagram representing IESs highly induced in the mutants. ( G ) A box plot comparing PDD1 enrichment in wild-type cells [(ChIP − input)/(ChIP + input)] in IESs induced in the mutants ( ΔDCL1 , ΔEZL1 , and ΔPDD1 cells, respectively) with IESs not induced in any mutants. The first quartile, median, and the third quartile are marked. A Kruskal-Wallis H test was performed for all three pairwise comparisons, revealing highly significant variances. P

    Article Snippet: The RNA samples, after oligo-dT enrichment of polyadenylated transcripts, were analyzed by strand-specific Illumina sequencing (RNA sequencing [RNA-seq]).

    Techniques: RNA Sequencing Assay, Chromatin Immunoprecipitation

    mRNA characteristics for IES-specific polyadenylated transcripts. ( A ) A representative GBrowse view illustrating strand bias and splice sites of IES-specific polyadenylated transcripts. Gene model: an hAT and a Tc1 family transposase. Positive values of y -axes represent normalized RNA-seq coverage (reads per million mapped reads, RPM) of the Watson strand, negative values the Crick strand. (Blue bar) IESs. ( B ) Venn diagrams representing splice sites associated with IES ( top ) and MDS ( bottom ) polyadenylated transcripts in wild type and mutants. ( C ) Strong strand bias for IES-specific polyadenylated transcripts. y -axis: Strand bias index, quantified by RNA-seq coverage of the Watson (W) and Crick strand (C) of a 200-bp bin: |W − C|/(W + C) higher value indicating stronger strand bias. x ). ( D ) Composite analysis of epigenetically silenced IES-specific loci. ( Top ). Each locus is equally divided into 100 units from the 5′ to 3′ end (highlighted region) and extended by 1.5 kb in both directions (the scale is not related to the gene body length). The average RPM of each unit in all the loci were cumulated. ( Inset ) Zoom-in of the highlighted area. Positive values of the y -axis represent the cumulative RPM from the sense strand of the predominant transcript (solid lines), negative values the antisense strand (dashed lines). ( Bottom ) Control with randomized genomic locations. Note the strong strand bias of IES-specific polyadenylated transcripts and their high induction in the mutants. Both characteristics disappeared in the randomized control. ( E ) A positive correlation between polyadenylated transcript levels and strand bias in IES-specific loci in ΔDCL1 cells. y -axis: Strand bias index, quantified by RNA-seq coverage of the sense (S) and antisense strand (A) of an IES-specific locus: (S − A)/(S + A); values close to 1 indicate strong bias for sense strand transcripts. x -axis: Quantiles of IES-specific loci ranked by their polyadenylated transcript levels in ΔDCL1 cells; all loci expressing polyadenylated transcripts are grouped into 10 quantiles (1–10, from low to high). ( F ) Codon usage patterns. IES-specific loci with long ORFs (≥100 amino acids) were searched for homology by blastp (e ≤ 1 × 10 −10 ). Codon usage patterns of IES-specific loci as well as regular genes encoded in MDS are plotted in a radar chart.

    Journal: Genes & Development

    Article Title: RNAi-dependent Polycomb repression controls transposable elements in Tetrahymena

    doi: 10.1101/gad.320796.118

    Figure Lengend Snippet: mRNA characteristics for IES-specific polyadenylated transcripts. ( A ) A representative GBrowse view illustrating strand bias and splice sites of IES-specific polyadenylated transcripts. Gene model: an hAT and a Tc1 family transposase. Positive values of y -axes represent normalized RNA-seq coverage (reads per million mapped reads, RPM) of the Watson strand, negative values the Crick strand. (Blue bar) IESs. ( B ) Venn diagrams representing splice sites associated with IES ( top ) and MDS ( bottom ) polyadenylated transcripts in wild type and mutants. ( C ) Strong strand bias for IES-specific polyadenylated transcripts. y -axis: Strand bias index, quantified by RNA-seq coverage of the Watson (W) and Crick strand (C) of a 200-bp bin: |W − C|/(W + C) higher value indicating stronger strand bias. x ). ( D ) Composite analysis of epigenetically silenced IES-specific loci. ( Top ). Each locus is equally divided into 100 units from the 5′ to 3′ end (highlighted region) and extended by 1.5 kb in both directions (the scale is not related to the gene body length). The average RPM of each unit in all the loci were cumulated. ( Inset ) Zoom-in of the highlighted area. Positive values of the y -axis represent the cumulative RPM from the sense strand of the predominant transcript (solid lines), negative values the antisense strand (dashed lines). ( Bottom ) Control with randomized genomic locations. Note the strong strand bias of IES-specific polyadenylated transcripts and their high induction in the mutants. Both characteristics disappeared in the randomized control. ( E ) A positive correlation between polyadenylated transcript levels and strand bias in IES-specific loci in ΔDCL1 cells. y -axis: Strand bias index, quantified by RNA-seq coverage of the sense (S) and antisense strand (A) of an IES-specific locus: (S − A)/(S + A); values close to 1 indicate strong bias for sense strand transcripts. x -axis: Quantiles of IES-specific loci ranked by their polyadenylated transcript levels in ΔDCL1 cells; all loci expressing polyadenylated transcripts are grouped into 10 quantiles (1–10, from low to high). ( F ) Codon usage patterns. IES-specific loci with long ORFs (≥100 amino acids) were searched for homology by blastp (e ≤ 1 × 10 −10 ). Codon usage patterns of IES-specific loci as well as regular genes encoded in MDS are plotted in a radar chart.

    Article Snippet: The RNA samples, after oligo-dT enrichment of polyadenylated transcripts, were analyzed by strand-specific Illumina sequencing (RNA sequencing [RNA-seq]).

    Techniques: HAT Assay, RNA Sequencing Assay, Expressing

    Transfection of non-phagocytic cells, using mRNA:LPNs and mRNA:CS-PLGA at different ratios performed in epithelial A549 cells for 24 h and 48 h post-transfection using flow cytometer. mRNA complexed LPNs reveal a significant higher transgene expression over CS-PLGA NPs. Both NPs show higher transfection rates with higher mRNA:NP ratios and increasing transfection until 48 h post-transfection N = 4, mean ± SD (*p

    Journal: Journal of Nanobiotechnology

    Article Title: Kinetics of mRNA delivery and protein translation in dendritic cells using lipid-coated PLGA nanoparticles

    doi: 10.1186/s12951-018-0401-y

    Figure Lengend Snippet: Transfection of non-phagocytic cells, using mRNA:LPNs and mRNA:CS-PLGA at different ratios performed in epithelial A549 cells for 24 h and 48 h post-transfection using flow cytometer. mRNA complexed LPNs reveal a significant higher transgene expression over CS-PLGA NPs. Both NPs show higher transfection rates with higher mRNA:NP ratios and increasing transfection until 48 h post-transfection N = 4, mean ± SD (*p

    Article Snippet: The encapsulation efficiency (%EE) of bound mRNA:LPNs and mRNA:CS-PLGA NPs was evaluated indirectly by pelleting all samples down at 24,400g for 30 min and determining the concentration of unbound mRNA in the supernatant by measuring absorbance at 260/280 nm with a NanoDrop Spectrophotometer.

    Techniques: Transfection, Flow Cytometry, Cytometry, Expressing

    a1 Representative images depicted from live cell video after nine different time-points. The images are part of a video provided in Additional file 2 : Movie S1. DC2.4 cells were incubated with mRNA:FA-LPNs for a complete time duration of 4 h and with an interval of 3 min/image. Green dots on the images represent the fluorescence signal of labeled LPNs, while the cells signaling in red are the ones with successful mCherry expression. Scale bar = 100 µm. a2 Time-dependent change of the red fluorescence signal resulting from transfected cells and non-transfected, which are correlated with a3 green fluorescence signal of labeled nanoparticles. The tendency of each single cell being transfected is independent of the NPs uptake, as no significant difference between the uptake-behavior of transfected and non-transfected cells was observable. λ em = peak emission wavelength, MFI mean fluorescence intensity (mean ± SD, data from n = 32 fluorescent cells, n = 8 non-fluorescent cells, obtained from 4 independent videos)

    Journal: Journal of Nanobiotechnology

    Article Title: Kinetics of mRNA delivery and protein translation in dendritic cells using lipid-coated PLGA nanoparticles

    doi: 10.1186/s12951-018-0401-y

    Figure Lengend Snippet: a1 Representative images depicted from live cell video after nine different time-points. The images are part of a video provided in Additional file 2 : Movie S1. DC2.4 cells were incubated with mRNA:FA-LPNs for a complete time duration of 4 h and with an interval of 3 min/image. Green dots on the images represent the fluorescence signal of labeled LPNs, while the cells signaling in red are the ones with successful mCherry expression. Scale bar = 100 µm. a2 Time-dependent change of the red fluorescence signal resulting from transfected cells and non-transfected, which are correlated with a3 green fluorescence signal of labeled nanoparticles. The tendency of each single cell being transfected is independent of the NPs uptake, as no significant difference between the uptake-behavior of transfected and non-transfected cells was observable. λ em = peak emission wavelength, MFI mean fluorescence intensity (mean ± SD, data from n = 32 fluorescent cells, n = 8 non-fluorescent cells, obtained from 4 independent videos)

    Article Snippet: The encapsulation efficiency (%EE) of bound mRNA:LPNs and mRNA:CS-PLGA NPs was evaluated indirectly by pelleting all samples down at 24,400g for 30 min and determining the concentration of unbound mRNA in the supernatant by measuring absorbance at 260/280 nm with a NanoDrop Spectrophotometer.

    Techniques: Incubation, Fluorescence, Labeling, Expressing, Transfection

    Representative confocal images of DC2.4 cells transfected with both mRNA complexed NPs and by using jetPRIME ® as a positive control, naked mRNA as negative control. Transfection was analyzed with CLSM a 24 h and b 48 h post-transfection. Red fluorescence reveals cells successfully transfected with the nanoparticles while their morphology remains consistent with non-transfected cells (staining: green: cell membrane; blue: cell nucleus; scale bar: 50 μm)

    Journal: Journal of Nanobiotechnology

    Article Title: Kinetics of mRNA delivery and protein translation in dendritic cells using lipid-coated PLGA nanoparticles

    doi: 10.1186/s12951-018-0401-y

    Figure Lengend Snippet: Representative confocal images of DC2.4 cells transfected with both mRNA complexed NPs and by using jetPRIME ® as a positive control, naked mRNA as negative control. Transfection was analyzed with CLSM a 24 h and b 48 h post-transfection. Red fluorescence reveals cells successfully transfected with the nanoparticles while their morphology remains consistent with non-transfected cells (staining: green: cell membrane; blue: cell nucleus; scale bar: 50 μm)

    Article Snippet: The encapsulation efficiency (%EE) of bound mRNA:LPNs and mRNA:CS-PLGA NPs was evaluated indirectly by pelleting all samples down at 24,400g for 30 min and determining the concentration of unbound mRNA in the supernatant by measuring absorbance at 260/280 nm with a NanoDrop Spectrophotometer.

    Techniques: Transfection, Positive Control, Negative Control, Confocal Laser Scanning Microscopy, Fluorescence, Staining

    Quantification of cellular NP association for fluoresceinamine (FA) labeled blank ( a1 , a2 ) and mRNA complexed nanoparticles ( b1 , b2 ) tested in DC2.4 cells. NPs were incubated for 2 h and 4 h at different concentrations (20, 40 and 60 µg/mL corresponding to the weight ratios 1:10, 1:20 and 1:30 used for transfection). a1 , b1 Green NP fluorescent signal quantified by flow cytometry. Blank LPNs tend to show more uptake then blank CS-PLGA NPs, whereas for mRNA-loaded nanoparticles the opposite was observed. None of the samples showed a significant difference between 2 and 4 h incubation. a2 , b2 Representative graphs obtained for NP samples after 4 h incubation. N = 4, mean ± SD

    Journal: Journal of Nanobiotechnology

    Article Title: Kinetics of mRNA delivery and protein translation in dendritic cells using lipid-coated PLGA nanoparticles

    doi: 10.1186/s12951-018-0401-y

    Figure Lengend Snippet: Quantification of cellular NP association for fluoresceinamine (FA) labeled blank ( a1 , a2 ) and mRNA complexed nanoparticles ( b1 , b2 ) tested in DC2.4 cells. NPs were incubated for 2 h and 4 h at different concentrations (20, 40 and 60 µg/mL corresponding to the weight ratios 1:10, 1:20 and 1:30 used for transfection). a1 , b1 Green NP fluorescent signal quantified by flow cytometry. Blank LPNs tend to show more uptake then blank CS-PLGA NPs, whereas for mRNA-loaded nanoparticles the opposite was observed. None of the samples showed a significant difference between 2 and 4 h incubation. a2 , b2 Representative graphs obtained for NP samples after 4 h incubation. N = 4, mean ± SD

    Article Snippet: The encapsulation efficiency (%EE) of bound mRNA:LPNs and mRNA:CS-PLGA NPs was evaluated indirectly by pelleting all samples down at 24,400g for 30 min and determining the concentration of unbound mRNA in the supernatant by measuring absorbance at 260/280 nm with a NanoDrop Spectrophotometer.

    Techniques: Labeling, Incubation, Transfection, Flow Cytometry, Cytometry

    The kinetics of transfection for both mRNA-loaded NPs was quantified using flow cytometry, which indicated a significant difference between a1 mRNA:LPNs over a2 mRNA:CS-PLGA NPs, while mRNA:LPNs with a ratio of 1:20 and 1:30 elucidated a significant higher transfection rate over 1:10. a3 Control samples for transfection studies were JetPRIME ® as positive control and naked mRNA as negative control. (Note the enlarged y-axis, N = 4, mean ± SD.) a4 Representative graphs for evaluated time-points demonstrate a strong fluorescence shift for mRNA:LPNs with an increment in time and hence a higher transgene expression

    Journal: Journal of Nanobiotechnology

    Article Title: Kinetics of mRNA delivery and protein translation in dendritic cells using lipid-coated PLGA nanoparticles

    doi: 10.1186/s12951-018-0401-y

    Figure Lengend Snippet: The kinetics of transfection for both mRNA-loaded NPs was quantified using flow cytometry, which indicated a significant difference between a1 mRNA:LPNs over a2 mRNA:CS-PLGA NPs, while mRNA:LPNs with a ratio of 1:20 and 1:30 elucidated a significant higher transfection rate over 1:10. a3 Control samples for transfection studies were JetPRIME ® as positive control and naked mRNA as negative control. (Note the enlarged y-axis, N = 4, mean ± SD.) a4 Representative graphs for evaluated time-points demonstrate a strong fluorescence shift for mRNA:LPNs with an increment in time and hence a higher transgene expression

    Article Snippet: The encapsulation efficiency (%EE) of bound mRNA:LPNs and mRNA:CS-PLGA NPs was evaluated indirectly by pelleting all samples down at 24,400g for 30 min and determining the concentration of unbound mRNA in the supernatant by measuring absorbance at 260/280 nm with a NanoDrop Spectrophotometer.

    Techniques: Transfection, Flow Cytometry, Cytometry, Positive Control, Negative Control, Fluorescence, Expressing

    Schematic illustration of all nanoparticles used in this study. Both, LPNs and CS-PLGA NPs either complexed with mRNA or/and labeled with fluoresceinamine are used to quantify their uptake behavior and transfection efficiency in a dendritic cell line (DC2.4)

    Journal: Journal of Nanobiotechnology

    Article Title: Kinetics of mRNA delivery and protein translation in dendritic cells using lipid-coated PLGA nanoparticles

    doi: 10.1186/s12951-018-0401-y

    Figure Lengend Snippet: Schematic illustration of all nanoparticles used in this study. Both, LPNs and CS-PLGA NPs either complexed with mRNA or/and labeled with fluoresceinamine are used to quantify their uptake behavior and transfection efficiency in a dendritic cell line (DC2.4)

    Article Snippet: The encapsulation efficiency (%EE) of bound mRNA:LPNs and mRNA:CS-PLGA NPs was evaluated indirectly by pelleting all samples down at 24,400g for 30 min and determining the concentration of unbound mRNA in the supernatant by measuring absorbance at 260/280 nm with a NanoDrop Spectrophotometer.

    Techniques: Labeling, Transfection

    Gel retardation assay of mRNA complexed with different nanoparticles (NPs) at various ratios: a LPNs and b CS-PLGA NPs. Both images indicate mRNA binding to NPs and their appropriate release using heparin

    Journal: Journal of Nanobiotechnology

    Article Title: Kinetics of mRNA delivery and protein translation in dendritic cells using lipid-coated PLGA nanoparticles

    doi: 10.1186/s12951-018-0401-y

    Figure Lengend Snippet: Gel retardation assay of mRNA complexed with different nanoparticles (NPs) at various ratios: a LPNs and b CS-PLGA NPs. Both images indicate mRNA binding to NPs and their appropriate release using heparin

    Article Snippet: The encapsulation efficiency (%EE) of bound mRNA:LPNs and mRNA:CS-PLGA NPs was evaluated indirectly by pelleting all samples down at 24,400g for 30 min and determining the concentration of unbound mRNA in the supernatant by measuring absorbance at 260/280 nm with a NanoDrop Spectrophotometer.

    Techniques: Electrophoretic Mobility Shift Assay, Binding Assay

    Morphology of mRNA-loaded LPNs ( a1 , a2 ) and CS-PLGA NPs ( b1 , b2 ) visualized using SEM and TEM. a1 , b1 SEM images show a smooth, spherical morphology of the mRNA-loaded nanoparticles and a2 , b2 TEM images show the core–shell structure of the particles after staining with 0.5% (w/V) PTA

    Journal: Journal of Nanobiotechnology

    Article Title: Kinetics of mRNA delivery and protein translation in dendritic cells using lipid-coated PLGA nanoparticles

    doi: 10.1186/s12951-018-0401-y

    Figure Lengend Snippet: Morphology of mRNA-loaded LPNs ( a1 , a2 ) and CS-PLGA NPs ( b1 , b2 ) visualized using SEM and TEM. a1 , b1 SEM images show a smooth, spherical morphology of the mRNA-loaded nanoparticles and a2 , b2 TEM images show the core–shell structure of the particles after staining with 0.5% (w/V) PTA

    Article Snippet: The encapsulation efficiency (%EE) of bound mRNA:LPNs and mRNA:CS-PLGA NPs was evaluated indirectly by pelleting all samples down at 24,400g for 30 min and determining the concentration of unbound mRNA in the supernatant by measuring absorbance at 260/280 nm with a NanoDrop Spectrophotometer.

    Techniques: Transmission Electron Microscopy, Staining

    Location of cto genes on the PRV genome. Both cto transcripts (CTOs) are polyadenylated RNAs with a common 3′ termination. CTO-L is generated by the continuation of transcription after the termination signals of the ul21 gene. OriL is the replication origin of in the UL region of viral DNA mapped between the ul21 and ul22 genes.

    Journal: Viruses

    Article Title: Characterization of Novel Transcripts in Pseudorabies Virus

    doi: 10.3390/v7052727

    Figure Lengend Snippet: Location of cto genes on the PRV genome. Both cto transcripts (CTOs) are polyadenylated RNAs with a common 3′ termination. CTO-L is generated by the continuation of transcription after the termination signals of the ul21 gene. OriL is the replication origin of in the UL region of viral DNA mapped between the ul21 and ul22 genes.

    Article Snippet: These polyadenylated RNAs were characterized by ascertaining their nucleotide sequences with the Illumina HiScanSQ and Pacific Biosciences Real-Time (PacBio RSII) sequencing platforms and by analyzing their transcription kinetics through use of multi-time-point Real-Time RT-PCR and the PacBio RSII system.

    Techniques: Generated

    Transcriptome profiling to explore the mechanism of the synthetic lethality. (A) and (B) REACTOME signal pathway analysis for the RNA sequencing results is shown. HCC1937 BRCA1 isogenic cells were treated with 5 μmol/L entinostat for 24 h and total RNA was extracted for RNA sequencing. The gene expression profiles were analyzed with REACTOME signal pathway database. The pathway genes that were significantly up- (A) and down-regulated (B) by entinostat are shown. (C) and (D) The effect of entinostat on cellular oxidative stress and DNA damage was examined. HCC1937 BRCA1 −/− (C) or T47D sh BRCA1 (D) cells were treated with 5 μmol/L entinostat for indicated time points and Western blots for proteins involved in oxidative stress and DNA damage responses were analyzed. GAPDH was used as an internal control. (E) RT-qPCR analysis for the TXNIP mRNA level was examined in HCC1937 BRCA1 isogenic cells treated with entinostat. (F) Chromatin immunoprecipitation (ChIP) of TXNIP promoter using anti-acetyl histone H4 antibody is shown. HCC1937 BRCA1 −/− cells were treated with 5 μmol/L entinostat for 6 h and processed for ChIP using a rabbit IgG (control) or anti-acetyl histone H4 antibody. The ChIP DNA was subjected to PCR amplification with a primer pair specific for TXNIP promoter. (G) and (I) The effect of HDAC inhibitors, entinostat (class I-HDACi, G), vorinostat (pan-HDACi, H), and mocetinostat (class I-HDACi, I) on histone acetylation, cellular oxidative stress and DNA damage responses was examined in HCC1937 BRCA1 isogenic cell lines. Data are mean ± SD, * P

    Journal: Acta Pharmaceutica Sinica. B

    Article Title: Class I histone deacetylase inhibition is synthetic lethal with BRCA1 deficiency in breast cancer cells

    doi: 10.1016/j.apsb.2019.08.008

    Figure Lengend Snippet: Transcriptome profiling to explore the mechanism of the synthetic lethality. (A) and (B) REACTOME signal pathway analysis for the RNA sequencing results is shown. HCC1937 BRCA1 isogenic cells were treated with 5 μmol/L entinostat for 24 h and total RNA was extracted for RNA sequencing. The gene expression profiles were analyzed with REACTOME signal pathway database. The pathway genes that were significantly up- (A) and down-regulated (B) by entinostat are shown. (C) and (D) The effect of entinostat on cellular oxidative stress and DNA damage was examined. HCC1937 BRCA1 −/− (C) or T47D sh BRCA1 (D) cells were treated with 5 μmol/L entinostat for indicated time points and Western blots for proteins involved in oxidative stress and DNA damage responses were analyzed. GAPDH was used as an internal control. (E) RT-qPCR analysis for the TXNIP mRNA level was examined in HCC1937 BRCA1 isogenic cells treated with entinostat. (F) Chromatin immunoprecipitation (ChIP) of TXNIP promoter using anti-acetyl histone H4 antibody is shown. HCC1937 BRCA1 −/− cells were treated with 5 μmol/L entinostat for 6 h and processed for ChIP using a rabbit IgG (control) or anti-acetyl histone H4 antibody. The ChIP DNA was subjected to PCR amplification with a primer pair specific for TXNIP promoter. (G) and (I) The effect of HDAC inhibitors, entinostat (class I-HDACi, G), vorinostat (pan-HDACi, H), and mocetinostat (class I-HDACi, I) on histone acetylation, cellular oxidative stress and DNA damage responses was examined in HCC1937 BRCA1 isogenic cell lines. Data are mean ± SD, * P

    Article Snippet: The RNA integrity number (RIN) values were assessed with RNA 6000 Nano Kit on 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) and were used for RNA quality evaluation. mRNA was extracted from the total RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490S, New England Biolabs, Ipswich, MA, USA). cDNA library was prepared from mRNA by NEBNext Ultra Directional RNA Library Prep Kit for Illumina (E7420S, New England Biolabs).

    Techniques: RNA Sequencing Assay, Expressing, Western Blot, Quantitative RT-PCR, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification

    The balance between ncRNA and mRNA production is a critical aspect of the balance between transcriptional silencing and activation. ( A ) IES-specific polyadenylated transcripts are only produced during developing MAC formation. (3) 3 h after mixing: meiosis, (6) 6 h after mixing: gametogenesis, (10) 10 h after mixing: developing MAC formation, (blue bar) IES. RNA-seq was performed after oligo-dT enrichment of polyadenylated transcripts. Note that essentially no RNA-seq reads were mapped to the IES region at the two early conjugation time points. (PM) Parental MAC, (DM) developing MAC, (OM) old MAC. ( B ) Percentage of RNA-seq reads mapped to consistently processed IESs, relative to total mappable reads. Note that IES-specific polyadenylated transcripts were abundantly produced during developing MAC formation (10 h after mixing) but rarely detected before that (3 and 6 h after mixing). ( C ) Localization of the transcriptional and cotranscriptional machineries in early (3 h after mixing) and late (10 h after mixing) conjugating cells. RPB3-HA, CBP20-HA, PRP19-HA, and THO2-HA cells were stained with an anti-HA antibody (red) and counterstained with DAPI (blue). Parental MAC (PM): green arrowhead, developing MAC (DM): green arrows, MIC: white arrowheads, old MAC (OM): white arrow. ( D ) Interaction between TWI1 and the transcriptional/cotranscriptional machineries. The designated cells were processed for crosslink-immunoprecipitation with the anti-HA antibody at late conjugation (10 h after mixing). The anti-HA and anti-TWI1 antibodies were used for immunoblotting. Note that similar amounts of bait proteins were recovered, as shown by the anti-HA immunoblotting. ( E ) Production of scnRNA from intronic as well as exonic regions. Distributions of polyadenylated transcripts (dotted lines) and scnRNA (solid lines) in introns and exons of IES-specific loci. Each intron is equally divided into 10 units from the 5′ to 3′ end; the two flanking exons are also equally divided into 10 units, respectively. The average RPM of each unit in all the loci were cumulated. Double counting for exons is avoided. ( F ) Alternative production of ncRNA and mRNA in the meiotic MIC and the developing MAC. In this simplified schematic, transcription from IES-specific loci is emphasized, while genic transcription from MDS is omitted. See text for details.

    Journal: Genes & Development

    Article Title: RNAi-dependent Polycomb repression controls transposable elements in Tetrahymena

    doi: 10.1101/gad.320796.118

    Figure Lengend Snippet: The balance between ncRNA and mRNA production is a critical aspect of the balance between transcriptional silencing and activation. ( A ) IES-specific polyadenylated transcripts are only produced during developing MAC formation. (3) 3 h after mixing: meiosis, (6) 6 h after mixing: gametogenesis, (10) 10 h after mixing: developing MAC formation, (blue bar) IES. RNA-seq was performed after oligo-dT enrichment of polyadenylated transcripts. Note that essentially no RNA-seq reads were mapped to the IES region at the two early conjugation time points. (PM) Parental MAC, (DM) developing MAC, (OM) old MAC. ( B ) Percentage of RNA-seq reads mapped to consistently processed IESs, relative to total mappable reads. Note that IES-specific polyadenylated transcripts were abundantly produced during developing MAC formation (10 h after mixing) but rarely detected before that (3 and 6 h after mixing). ( C ) Localization of the transcriptional and cotranscriptional machineries in early (3 h after mixing) and late (10 h after mixing) conjugating cells. RPB3-HA, CBP20-HA, PRP19-HA, and THO2-HA cells were stained with an anti-HA antibody (red) and counterstained with DAPI (blue). Parental MAC (PM): green arrowhead, developing MAC (DM): green arrows, MIC: white arrowheads, old MAC (OM): white arrow. ( D ) Interaction between TWI1 and the transcriptional/cotranscriptional machineries. The designated cells were processed for crosslink-immunoprecipitation with the anti-HA antibody at late conjugation (10 h after mixing). The anti-HA and anti-TWI1 antibodies were used for immunoblotting. Note that similar amounts of bait proteins were recovered, as shown by the anti-HA immunoblotting. ( E ) Production of scnRNA from intronic as well as exonic regions. Distributions of polyadenylated transcripts (dotted lines) and scnRNA (solid lines) in introns and exons of IES-specific loci. Each intron is equally divided into 10 units from the 5′ to 3′ end; the two flanking exons are also equally divided into 10 units, respectively. The average RPM of each unit in all the loci were cumulated. Double counting for exons is avoided. ( F ) Alternative production of ncRNA and mRNA in the meiotic MIC and the developing MAC. In this simplified schematic, transcription from IES-specific loci is emphasized, while genic transcription from MDS is omitted. See text for details.

    Article Snippet: Polyadenylated transcripts were enriched using Sera-Mag magnetic oligo-dT beads (GE).

    Techniques: Activation Assay, Produced, RNA Sequencing Assay, Conjugation Assay, Staining, Immunoprecipitation

    Widespread production of IES-specific polyadenylated transcripts in mutants deficient in RNAi-dependent Polycomb repression. ( A ) A schematic for DNA elimination in Tetrahymena . ( B ) Key steps and players in the DNA elimination pathway. ( C ) from highest to lowest by their coverage of polyadenylated RNA (reads per kilobase of transcript per million mapped reads [RPKM]), in wild type and mutants deficient in RNAi-dependent Polycomb repression ( ΔDCL1, ΔEZL1 , and ΔPDD1 , respectively). ( Inset ) The top 100 IESs. Note increased and widespread transcription of IES-specific sequences in the mutants. ( D ) Distribution of highly expressed IESs, in wild type and the mutants. Supercontig 2.1, over 3.5 Mb in length, is analyzed. Read densities (RPKM) are represented by a color scale. Note the alignment of red stripes in the mutants, indicating coregulation by the RNAi-dependent Polycomb repression pathway. ( E ) A representative GBrowse view illustrating RNA sequencing (RNA-seq) coverage in wild type and the mutants. (Blue bar) IES. Gene model: an IES-specific ( left ) and an MDS-specific ( right ) gene. Note dramatically increased IES transcription in the mutants, compared with wild-type cells. ( F ) A proportional Venn diagram representing IESs highly induced in the mutants. ( G ) A box plot comparing PDD1 enrichment in wild-type cells [(ChIP − input)/(ChIP + input)] in IESs induced in the mutants ( ΔDCL1 , ΔEZL1 , and ΔPDD1 cells, respectively) with IESs not induced in any mutants. The first quartile, median, and the third quartile are marked. A Kruskal-Wallis H test was performed for all three pairwise comparisons, revealing highly significant variances. P

    Journal: Genes & Development

    Article Title: RNAi-dependent Polycomb repression controls transposable elements in Tetrahymena

    doi: 10.1101/gad.320796.118

    Figure Lengend Snippet: Widespread production of IES-specific polyadenylated transcripts in mutants deficient in RNAi-dependent Polycomb repression. ( A ) A schematic for DNA elimination in Tetrahymena . ( B ) Key steps and players in the DNA elimination pathway. ( C ) from highest to lowest by their coverage of polyadenylated RNA (reads per kilobase of transcript per million mapped reads [RPKM]), in wild type and mutants deficient in RNAi-dependent Polycomb repression ( ΔDCL1, ΔEZL1 , and ΔPDD1 , respectively). ( Inset ) The top 100 IESs. Note increased and widespread transcription of IES-specific sequences in the mutants. ( D ) Distribution of highly expressed IESs, in wild type and the mutants. Supercontig 2.1, over 3.5 Mb in length, is analyzed. Read densities (RPKM) are represented by a color scale. Note the alignment of red stripes in the mutants, indicating coregulation by the RNAi-dependent Polycomb repression pathway. ( E ) A representative GBrowse view illustrating RNA sequencing (RNA-seq) coverage in wild type and the mutants. (Blue bar) IES. Gene model: an IES-specific ( left ) and an MDS-specific ( right ) gene. Note dramatically increased IES transcription in the mutants, compared with wild-type cells. ( F ) A proportional Venn diagram representing IESs highly induced in the mutants. ( G ) A box plot comparing PDD1 enrichment in wild-type cells [(ChIP − input)/(ChIP + input)] in IESs induced in the mutants ( ΔDCL1 , ΔEZL1 , and ΔPDD1 cells, respectively) with IESs not induced in any mutants. The first quartile, median, and the third quartile are marked. A Kruskal-Wallis H test was performed for all three pairwise comparisons, revealing highly significant variances. P

    Article Snippet: Polyadenylated transcripts were enriched using Sera-Mag magnetic oligo-dT beads (GE).

    Techniques: RNA Sequencing Assay, Chromatin Immunoprecipitation

    mRNA characteristics for IES-specific polyadenylated transcripts. ( A ) A representative GBrowse view illustrating strand bias and splice sites of IES-specific polyadenylated transcripts. Gene model: an hAT and a Tc1 family transposase. Positive values of y -axes represent normalized RNA-seq coverage (reads per million mapped reads, RPM) of the Watson strand, negative values the Crick strand. (Blue bar) IESs. ( B ) Venn diagrams representing splice sites associated with IES ( top ) and MDS ( bottom ) polyadenylated transcripts in wild type and mutants. ( C ) Strong strand bias for IES-specific polyadenylated transcripts. y -axis: Strand bias index, quantified by RNA-seq coverage of the Watson (W) and Crick strand (C) of a 200-bp bin: |W − C|/(W + C) higher value indicating stronger strand bias. x ). ( D ) Composite analysis of epigenetically silenced IES-specific loci. ( Top ). Each locus is equally divided into 100 units from the 5′ to 3′ end (highlighted region) and extended by 1.5 kb in both directions (the scale is not related to the gene body length). The average RPM of each unit in all the loci were cumulated. ( Inset ) Zoom-in of the highlighted area. Positive values of the y -axis represent the cumulative RPM from the sense strand of the predominant transcript (solid lines), negative values the antisense strand (dashed lines). ( Bottom ) Control with randomized genomic locations. Note the strong strand bias of IES-specific polyadenylated transcripts and their high induction in the mutants. Both characteristics disappeared in the randomized control. ( E ) A positive correlation between polyadenylated transcript levels and strand bias in IES-specific loci in ΔDCL1 cells. y -axis: Strand bias index, quantified by RNA-seq coverage of the sense (S) and antisense strand (A) of an IES-specific locus: (S − A)/(S + A); values close to 1 indicate strong bias for sense strand transcripts. x -axis: Quantiles of IES-specific loci ranked by their polyadenylated transcript levels in ΔDCL1 cells; all loci expressing polyadenylated transcripts are grouped into 10 quantiles (1–10, from low to high). ( F ) Codon usage patterns. IES-specific loci with long ORFs (≥100 amino acids) were searched for homology by blastp (e ≤ 1 × 10 −10 ). Codon usage patterns of IES-specific loci as well as regular genes encoded in MDS are plotted in a radar chart.

    Journal: Genes & Development

    Article Title: RNAi-dependent Polycomb repression controls transposable elements in Tetrahymena

    doi: 10.1101/gad.320796.118

    Figure Lengend Snippet: mRNA characteristics for IES-specific polyadenylated transcripts. ( A ) A representative GBrowse view illustrating strand bias and splice sites of IES-specific polyadenylated transcripts. Gene model: an hAT and a Tc1 family transposase. Positive values of y -axes represent normalized RNA-seq coverage (reads per million mapped reads, RPM) of the Watson strand, negative values the Crick strand. (Blue bar) IESs. ( B ) Venn diagrams representing splice sites associated with IES ( top ) and MDS ( bottom ) polyadenylated transcripts in wild type and mutants. ( C ) Strong strand bias for IES-specific polyadenylated transcripts. y -axis: Strand bias index, quantified by RNA-seq coverage of the Watson (W) and Crick strand (C) of a 200-bp bin: |W − C|/(W + C) higher value indicating stronger strand bias. x ). ( D ) Composite analysis of epigenetically silenced IES-specific loci. ( Top ). Each locus is equally divided into 100 units from the 5′ to 3′ end (highlighted region) and extended by 1.5 kb in both directions (the scale is not related to the gene body length). The average RPM of each unit in all the loci were cumulated. ( Inset ) Zoom-in of the highlighted area. Positive values of the y -axis represent the cumulative RPM from the sense strand of the predominant transcript (solid lines), negative values the antisense strand (dashed lines). ( Bottom ) Control with randomized genomic locations. Note the strong strand bias of IES-specific polyadenylated transcripts and their high induction in the mutants. Both characteristics disappeared in the randomized control. ( E ) A positive correlation between polyadenylated transcript levels and strand bias in IES-specific loci in ΔDCL1 cells. y -axis: Strand bias index, quantified by RNA-seq coverage of the sense (S) and antisense strand (A) of an IES-specific locus: (S − A)/(S + A); values close to 1 indicate strong bias for sense strand transcripts. x -axis: Quantiles of IES-specific loci ranked by their polyadenylated transcript levels in ΔDCL1 cells; all loci expressing polyadenylated transcripts are grouped into 10 quantiles (1–10, from low to high). ( F ) Codon usage patterns. IES-specific loci with long ORFs (≥100 amino acids) were searched for homology by blastp (e ≤ 1 × 10 −10 ). Codon usage patterns of IES-specific loci as well as regular genes encoded in MDS are plotted in a radar chart.

    Article Snippet: Polyadenylated transcripts were enriched using Sera-Mag magnetic oligo-dT beads (GE).

    Techniques: HAT Assay, RNA Sequencing Assay, Expressing

    Stimulation of HIEC/IEC-6 cells with LPS induced TNFα, as demonstrated by RT-PCR (A). Already after a stimulation period of three hours TNFα mRNA transcripts were observed in both IEC models. The expression of G3PDH was used as house keeping gene. The translation into the protein product and subsequent secretion of TNFα into the culture medium was analysed in HIEC using a high sensitivity ELISA (B). TNFα was produced in a dose dependent manner after stimulation with LPS. CHX treatment completely suppressed the secretion of TNFα. In keeping, functional experiments with CHX showed in HIEC that the proliferation rate after LPS stimulation remained unchanged once TNFα production was suppressed (C). On the other hand, addition of TNFα in the presence of CHX was still able to suppress HIEC proliferation. Concentrations (x axis) for CHX or LPS in μg/ml, for TNFα in ng/ml. The concentrations of CHX used together with LPS or TNFα were 1 μg/ml.

    Journal: Gut

    Article Title: Lipopolysaccharide modulation of normal enterocyte turnover by toll-like receptors is mediated by endogenously produced tumour necrosis factor ?

    doi:

    Figure Lengend Snippet: Stimulation of HIEC/IEC-6 cells with LPS induced TNFα, as demonstrated by RT-PCR (A). Already after a stimulation period of three hours TNFα mRNA transcripts were observed in both IEC models. The expression of G3PDH was used as house keeping gene. The translation into the protein product and subsequent secretion of TNFα into the culture medium was analysed in HIEC using a high sensitivity ELISA (B). TNFα was produced in a dose dependent manner after stimulation with LPS. CHX treatment completely suppressed the secretion of TNFα. In keeping, functional experiments with CHX showed in HIEC that the proliferation rate after LPS stimulation remained unchanged once TNFα production was suppressed (C). On the other hand, addition of TNFα in the presence of CHX was still able to suppress HIEC proliferation. Concentrations (x axis) for CHX or LPS in μg/ml, for TNFα in ng/ml. The concentrations of CHX used together with LPS or TNFα were 1 μg/ml.

    Article Snippet: mRNA was isolated from HIEC and IEC-6 cells at different time points (one to nine hours) after LPS stimulation using a Quickprep mRNA micro purification kit (Amersham Pharmacia Biotech, Freiburg, Germany).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, Produced, Functional Assay

    TLR expression in HIEC: RT-PCR analysis clearly showed TLR2 and TLR4 mRNA transcripts in unstimulated HIEC as well as Caco-2 and HT29 cells (A). Subsequent isolation and sequencing confirmed the identity of TLR2 and TLR4. In addition, all cell lines expressed MD2, a costimulatory molecule associated with TLR4. Western blot analysis (B) confirmed that HIEC express TLR2 (86 kDa) as well as TLR4 (88 kDa). Caco-2 cells served as positive control. In addition, immunofluorescence analysis (C) revealed a clear signal of membrane expressed TLR2 and TLR4 on native HIEC. The regulation of the expression of TLR2 and TLR4 by LPS was analysed by flow cytometry (D). TLR4 expression was higher in unstimulated HIEC compared with TLR2. LPS (10 μg/ml, 48 hours) significantly reduced TLR4 expression without changing TLR2 expression in HIEC. One of three representative experiments is shown.

    Journal: Gut

    Article Title: Lipopolysaccharide modulation of normal enterocyte turnover by toll-like receptors is mediated by endogenously produced tumour necrosis factor ?

    doi:

    Figure Lengend Snippet: TLR expression in HIEC: RT-PCR analysis clearly showed TLR2 and TLR4 mRNA transcripts in unstimulated HIEC as well as Caco-2 and HT29 cells (A). Subsequent isolation and sequencing confirmed the identity of TLR2 and TLR4. In addition, all cell lines expressed MD2, a costimulatory molecule associated with TLR4. Western blot analysis (B) confirmed that HIEC express TLR2 (86 kDa) as well as TLR4 (88 kDa). Caco-2 cells served as positive control. In addition, immunofluorescence analysis (C) revealed a clear signal of membrane expressed TLR2 and TLR4 on native HIEC. The regulation of the expression of TLR2 and TLR4 by LPS was analysed by flow cytometry (D). TLR4 expression was higher in unstimulated HIEC compared with TLR2. LPS (10 μg/ml, 48 hours) significantly reduced TLR4 expression without changing TLR2 expression in HIEC. One of three representative experiments is shown.

    Article Snippet: mRNA was isolated from HIEC and IEC-6 cells at different time points (one to nine hours) after LPS stimulation using a Quickprep mRNA micro purification kit (Amersham Pharmacia Biotech, Freiburg, Germany).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Sequencing, Western Blot, Positive Control, Immunofluorescence, Flow Cytometry, Cytometry

    microHIVE differentiation of spinal motor neurons. (A) Target and experimental molecular profiles of retinoic acid and GDF11. (Left) Based on correlative analysis of growth factor concentrations and RNA profiling, we designed a continuous profile of retinoic acid and GDF11 to induce rostral-caudal patterning of motor neurons, to correspond to the brachial, thoracic and lumbar regions of the spinal cord. (Right) The microHIVE experimental profile showed a close correlation to the desired target profile (profile similarity metric, ε = 0.991). (B) Schematic illustration of chamber binning. After motor neuron differentiation (day 28), the all-polymer cell culture chamber could be sectioned into seven bins for cellular characterization and spatial correlation. (C) Differential expressions of HOX genes across the chamber bins. HOX gene expressions were profiled from the respective chamber sections through quantitative PCR. All gene expression analyses were made relative to intrinsic GAPDH mRNA levels, and subsequently gene (column) normalized across all bins to compare respective gene expression trends in the form of a heat map. (D) Immunofluorescence confirmation of HOX protein expressions. Immunostaining of HOXB4 and HOXC8 (red) and SMI-32 (green) across the seven culture bins demonstrated successful generation of rostral (HOXB4) to caudal (HOXC8) motor neurons. Cellular nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. Cellular quantification based on positive staining of HOXB4 and HOXC8 further confirmed the respective protein expression trends. All measurements were performed in triplicate, and the data are displayed as mean ± s.d. in A and D .

    Journal: Theranostics

    Article Title: Microhexagon gradient array directs spatial diversification of spinal motor neurons

    doi: 10.7150/thno.29755

    Figure Lengend Snippet: microHIVE differentiation of spinal motor neurons. (A) Target and experimental molecular profiles of retinoic acid and GDF11. (Left) Based on correlative analysis of growth factor concentrations and RNA profiling, we designed a continuous profile of retinoic acid and GDF11 to induce rostral-caudal patterning of motor neurons, to correspond to the brachial, thoracic and lumbar regions of the spinal cord. (Right) The microHIVE experimental profile showed a close correlation to the desired target profile (profile similarity metric, ε = 0.991). (B) Schematic illustration of chamber binning. After motor neuron differentiation (day 28), the all-polymer cell culture chamber could be sectioned into seven bins for cellular characterization and spatial correlation. (C) Differential expressions of HOX genes across the chamber bins. HOX gene expressions were profiled from the respective chamber sections through quantitative PCR. All gene expression analyses were made relative to intrinsic GAPDH mRNA levels, and subsequently gene (column) normalized across all bins to compare respective gene expression trends in the form of a heat map. (D) Immunofluorescence confirmation of HOX protein expressions. Immunostaining of HOXB4 and HOXC8 (red) and SMI-32 (green) across the seven culture bins demonstrated successful generation of rostral (HOXB4) to caudal (HOXC8) motor neurons. Cellular nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. Cellular quantification based on positive staining of HOXB4 and HOXC8 further confirmed the respective protein expression trends. All measurements were performed in triplicate, and the data are displayed as mean ± s.d. in A and D .

    Article Snippet: For bin-based RNA sequencing, polyA enrichment of mRNA was performed for Illumina sequencing library preparation, according to the manufacturer's kit instructions with standard index sequences (Illumina).

    Techniques: Cell Culture, Real-time Polymerase Chain Reaction, Expressing, Immunofluorescence, Immunostaining, Staining

    Derivation of spinal motor neurons from human pluripotent stem cells. (A) Schematic illustration of spinal motor neuron differentiation from induced pluripotent stem cell (iPSC). Varying concentrations of retinoic acid and GDF11 were used to generate thoracic/lumbar motor neurons. (B) Immunofluorescence analysis of cellular identities. (Top) Co-staining of Nestin (green) and SOX1 (red) confirmed the generation of neural progenitor cells (NPC) at day 10 of cellular differentiation. (Bottom) Co-staining of SMI-32 (green) and ISL1 (red) at day 28 demonstrated successful differentiation of iPSCs into spinal motor neurons (MN). Cellular nuclei were counterstained with DAPI (blue). All scale bars indicate 50 μm. (C) RNA confirmation of cellular identities. mRNA expression levels of the corresponding neural progenitor and motor neuron markers were measured by quantitative PCR. All gene expression analyses were normalized to that of iPSC mRNA levels. (**** P

    Journal: Theranostics

    Article Title: Microhexagon gradient array directs spatial diversification of spinal motor neurons

    doi: 10.7150/thno.29755

    Figure Lengend Snippet: Derivation of spinal motor neurons from human pluripotent stem cells. (A) Schematic illustration of spinal motor neuron differentiation from induced pluripotent stem cell (iPSC). Varying concentrations of retinoic acid and GDF11 were used to generate thoracic/lumbar motor neurons. (B) Immunofluorescence analysis of cellular identities. (Top) Co-staining of Nestin (green) and SOX1 (red) confirmed the generation of neural progenitor cells (NPC) at day 10 of cellular differentiation. (Bottom) Co-staining of SMI-32 (green) and ISL1 (red) at day 28 demonstrated successful differentiation of iPSCs into spinal motor neurons (MN). Cellular nuclei were counterstained with DAPI (blue). All scale bars indicate 50 μm. (C) RNA confirmation of cellular identities. mRNA expression levels of the corresponding neural progenitor and motor neuron markers were measured by quantitative PCR. All gene expression analyses were normalized to that of iPSC mRNA levels. (**** P

    Article Snippet: For bin-based RNA sequencing, polyA enrichment of mRNA was performed for Illumina sequencing library preparation, according to the manufacturer's kit instructions with standard index sequences (Illumina).

    Techniques: Immunofluorescence, Staining, Cell Differentiation, Expressing, Real-time Polymerase Chain Reaction