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Illumina Inc ultra directional rna library prep kit
POU5F1 expression is stably sustained in <t>MYOD1-mRNA</t> (synMYOD1)-treated hESCs. ( a ) synMYOD1 was synthesized in vitro with T7 <t>RNA</t> polymerase. The template cDNA was flanked by 5′UTR and 3′UTR of alpha-globin with an oligo(T) 120 for adding a polyA tail. ARCA (5′cap analog), pseudo-UTP, and 5-methyl-CTP were incorporated to increase mRNA stability and translation efficiency. ( b ) The percentage of mRNA transfection in hESCs was tested using synthetic mRNA encoding Emerald GFP by FACS analysis. ( c ) Schematic diagram of the transfection protocol. hESCs were transfected with synMYOD1 once on day 0, twice on day 1, and once on day 2. ( d ) Immunostaining analysis for MyHC in the synMYOD1-transfected cells. Nuclei were stained with DAPI. The percentage of MyHC-stained cells is shown (mean ± SEM from four independent biological replicates). Scale bar: 200 μm. ( e ) Immunostaining analysis for POU5F1 in the synMYOD1-transfected cells at day 0 to day 3 post transfection. MYOD1 was detected by a MYOD1 specific antibody. Nuclei were stained with DAPI. Scale bar: 10 μm. ( f ) Immunostaining analysis for NANOG in the synMYOD1-transfected cells at day 0 to day 3 post transfection. MYOD1 was detected by the specific antibody. Nuclei were stained with DAPI. Scale bar: 10 μm. ( g ) qRT-PCR analysis for POU5F1 expression day 0 to day 3 after transfection (mean ± SEM from two independent biological replicates). ( h .
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1) Product Images from "Efficient differentiation of human pluripotent stem cells into skeletal muscle cells by combining RNA-based MYOD1-expression and POU5F1-silencing"

Article Title: Efficient differentiation of human pluripotent stem cells into skeletal muscle cells by combining RNA-based MYOD1-expression and POU5F1-silencing

Journal: Scientific Reports

doi: 10.1038/s41598-017-19114-y

POU5F1 expression is stably sustained in MYOD1-mRNA (synMYOD1)-treated hESCs. ( a ) synMYOD1 was synthesized in vitro with T7 RNA polymerase. The template cDNA was flanked by 5′UTR and 3′UTR of alpha-globin with an oligo(T) 120 for adding a polyA tail. ARCA (5′cap analog), pseudo-UTP, and 5-methyl-CTP were incorporated to increase mRNA stability and translation efficiency. ( b ) The percentage of mRNA transfection in hESCs was tested using synthetic mRNA encoding Emerald GFP by FACS analysis. ( c ) Schematic diagram of the transfection protocol. hESCs were transfected with synMYOD1 once on day 0, twice on day 1, and once on day 2. ( d ) Immunostaining analysis for MyHC in the synMYOD1-transfected cells. Nuclei were stained with DAPI. The percentage of MyHC-stained cells is shown (mean ± SEM from four independent biological replicates). Scale bar: 200 μm. ( e ) Immunostaining analysis for POU5F1 in the synMYOD1-transfected cells at day 0 to day 3 post transfection. MYOD1 was detected by a MYOD1 specific antibody. Nuclei were stained with DAPI. Scale bar: 10 μm. ( f ) Immunostaining analysis for NANOG in the synMYOD1-transfected cells at day 0 to day 3 post transfection. MYOD1 was detected by the specific antibody. Nuclei were stained with DAPI. Scale bar: 10 μm. ( g ) qRT-PCR analysis for POU5F1 expression day 0 to day 3 after transfection (mean ± SEM from two independent biological replicates). ( h .
Figure Legend Snippet: POU5F1 expression is stably sustained in MYOD1-mRNA (synMYOD1)-treated hESCs. ( a ) synMYOD1 was synthesized in vitro with T7 RNA polymerase. The template cDNA was flanked by 5′UTR and 3′UTR of alpha-globin with an oligo(T) 120 for adding a polyA tail. ARCA (5′cap analog), pseudo-UTP, and 5-methyl-CTP were incorporated to increase mRNA stability and translation efficiency. ( b ) The percentage of mRNA transfection in hESCs was tested using synthetic mRNA encoding Emerald GFP by FACS analysis. ( c ) Schematic diagram of the transfection protocol. hESCs were transfected with synMYOD1 once on day 0, twice on day 1, and once on day 2. ( d ) Immunostaining analysis for MyHC in the synMYOD1-transfected cells. Nuclei were stained with DAPI. The percentage of MyHC-stained cells is shown (mean ± SEM from four independent biological replicates). Scale bar: 200 μm. ( e ) Immunostaining analysis for POU5F1 in the synMYOD1-transfected cells at day 0 to day 3 post transfection. MYOD1 was detected by a MYOD1 specific antibody. Nuclei were stained with DAPI. Scale bar: 10 μm. ( f ) Immunostaining analysis for NANOG in the synMYOD1-transfected cells at day 0 to day 3 post transfection. MYOD1 was detected by the specific antibody. Nuclei were stained with DAPI. Scale bar: 10 μm. ( g ) qRT-PCR analysis for POU5F1 expression day 0 to day 3 after transfection (mean ± SEM from two independent biological replicates). ( h .

Techniques Used: Expressing, Stable Transfection, Synthesized, In Vitro, Transfection, FACS, Immunostaining, Staining, Quantitative RT-PCR

2) Product Images from "Chloride intracellular channel 1 (CLIC1) contributes to modulation of cyclic AMP‐activated whole‐cell chloride currents in human bronchial epithelial cells. Chloride intracellular channel 1 (CLIC1) contributes to modulation of cyclic AMP‐activated whole cell chloride currents in human bronchial epithelial cells"

Article Title: Chloride intracellular channel 1 (CLIC1) contributes to modulation of cyclic AMP‐activated whole‐cell chloride currents in human bronchial epithelial cells. Chloride intracellular channel 1 (CLIC1) contributes to modulation of cyclic AMP‐activated whole cell chloride currents in human bronchial epithelial cells

Journal: Physiological Reports

doi: 10.14814/phy2.13508

Transfection of si RNA specifically targeted against CLIC 1 significantly reduces both mRNA and protein expressions of CLIC 1 in cultured hBEC cells . Forty‐eight hours posttransfection of CLIC 1‐targeted si RNA (1 nmol/L) both mRNA (duplicate n = 3) (A) and protein (B and C) expression of CLIC 1 was reduced in cultured hBEC cells (B is a representative image of n = 6, **** denotes P
Figure Legend Snippet: Transfection of si RNA specifically targeted against CLIC 1 significantly reduces both mRNA and protein expressions of CLIC 1 in cultured hBEC cells . Forty‐eight hours posttransfection of CLIC 1‐targeted si RNA (1 nmol/L) both mRNA (duplicate n = 3) (A) and protein (B and C) expression of CLIC 1 was reduced in cultured hBEC cells (B is a representative image of n = 6, **** denotes P

Techniques Used: Transfection, Cell Culture, Expressing

3) Product Images from "Efficient differentiation of human pluripotent stem cells into skeletal muscle cells by combining RNA-based MYOD1-expression and POU5F1-silencing"

Article Title: Efficient differentiation of human pluripotent stem cells into skeletal muscle cells by combining RNA-based MYOD1-expression and POU5F1-silencing

Journal: Scientific Reports

doi: 10.1038/s41598-017-19114-y

POU5F1 expression is stably sustained in MYOD1-mRNA (synMYOD1)-treated hESCs. ( a ) synMYOD1 was synthesized in vitro with T7 RNA polymerase. The template cDNA was flanked by 5′UTR and 3′UTR of alpha-globin with an oligo(T) 120 for adding a polyA tail. ARCA (5′cap analog), pseudo-UTP, and 5-methyl-CTP were incorporated to increase mRNA stability and translation efficiency. ( b ) The percentage of mRNA transfection in hESCs was tested using synthetic mRNA encoding Emerald GFP by FACS analysis. ( c ) Schematic diagram of the transfection protocol. hESCs were transfected with synMYOD1 once on day 0, twice on day 1, and once on day 2. ( d ) Immunostaining analysis for MyHC in the synMYOD1-transfected cells. Nuclei were stained with DAPI. The percentage of MyHC-stained cells is shown (mean ± SEM from four independent biological replicates). Scale bar: 200 μm. ( e ) Immunostaining analysis for POU5F1 in the synMYOD1-transfected cells at day 0 to day 3 post transfection. MYOD1 was detected by a MYOD1 specific antibody. Nuclei were stained with DAPI. Scale bar: 10 μm. ( f ) Immunostaining analysis for NANOG in the synMYOD1-transfected cells at day 0 to day 3 post transfection. MYOD1 was detected by the specific antibody. Nuclei were stained with DAPI. Scale bar: 10 μm. ( g ) qRT-PCR analysis for POU5F1 expression day 0 to day 3 after transfection (mean ± SEM from two independent biological replicates). ( h ) Immunoblotting analysis for POU5F1 in the synMYOD1-transfected cells at day 3 post transfection. MYOD1 was detected by the specific antibody. The H3 antibody was used as a loading control. The relative intensities of POU5F1 signals normalized by H3 were compared between no transfection and synMYOD1 transfection (mean ± SEM from three independent biological replicates). NS: not significant. Uncropped images of the blots for Fig. 1h are shown in Supplementary Figure 7 .
Figure Legend Snippet: POU5F1 expression is stably sustained in MYOD1-mRNA (synMYOD1)-treated hESCs. ( a ) synMYOD1 was synthesized in vitro with T7 RNA polymerase. The template cDNA was flanked by 5′UTR and 3′UTR of alpha-globin with an oligo(T) 120 for adding a polyA tail. ARCA (5′cap analog), pseudo-UTP, and 5-methyl-CTP were incorporated to increase mRNA stability and translation efficiency. ( b ) The percentage of mRNA transfection in hESCs was tested using synthetic mRNA encoding Emerald GFP by FACS analysis. ( c ) Schematic diagram of the transfection protocol. hESCs were transfected with synMYOD1 once on day 0, twice on day 1, and once on day 2. ( d ) Immunostaining analysis for MyHC in the synMYOD1-transfected cells. Nuclei were stained with DAPI. The percentage of MyHC-stained cells is shown (mean ± SEM from four independent biological replicates). Scale bar: 200 μm. ( e ) Immunostaining analysis for POU5F1 in the synMYOD1-transfected cells at day 0 to day 3 post transfection. MYOD1 was detected by a MYOD1 specific antibody. Nuclei were stained with DAPI. Scale bar: 10 μm. ( f ) Immunostaining analysis for NANOG in the synMYOD1-transfected cells at day 0 to day 3 post transfection. MYOD1 was detected by the specific antibody. Nuclei were stained with DAPI. Scale bar: 10 μm. ( g ) qRT-PCR analysis for POU5F1 expression day 0 to day 3 after transfection (mean ± SEM from two independent biological replicates). ( h ) Immunoblotting analysis for POU5F1 in the synMYOD1-transfected cells at day 3 post transfection. MYOD1 was detected by the specific antibody. The H3 antibody was used as a loading control. The relative intensities of POU5F1 signals normalized by H3 were compared between no transfection and synMYOD1 transfection (mean ± SEM from three independent biological replicates). NS: not significant. Uncropped images of the blots for Fig. 1h are shown in Supplementary Figure 7 .

Techniques Used: Expressing, Stable Transfection, Synthesized, In Vitro, Transfection, FACS, Immunostaining, Staining, Quantitative RT-PCR

Related Articles

Electrophoresis:

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Multiplex Assay:

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Amplification:

Article Title: Chloride intracellular channel 1 (CLIC1) contributes to modulation of cyclic AMP‐activated whole‐cell chloride currents in human bronchial epithelial cells. Chloride intracellular channel 1 (CLIC1) contributes to modulation of cyclic AMP‐activated whole cell chloride currents in human bronchial epithelial cells
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RNA Sequencing Assay:

Article Title: Efficient differentiation of human pluripotent stem cells into skeletal muscle cells by combining RNA-based MYOD1-expression and POU5F1-silencing
Article Snippet: .. RNA-sequencing The cDNA libraries of siControl-treated cells, siPOU5F1-treated cells, and siPOU5F1/synMYOD1-treated cells were prepared from 500 ng of each total RNA sample for massive parallel sequencing using an NEBNext Poly(A) mRNA Magnetic Isolation Module and an Ultra Directional RNA Library Prep Kit for Illumina (NEB). .. The cDNA library produced ranged from 400 to 1000 bp, including the adaptor sequences.

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Article Title: Chloride intracellular channel 1 (CLIC1) contributes to modulation of cyclic AMP‐activated whole‐cell chloride currents in human bronchial epithelial cells. Chloride intracellular channel 1 (CLIC1) contributes to modulation of cyclic AMP‐activated whole cell chloride currents in human bronchial epithelial cells
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Article Title: Efficient differentiation of human pluripotent stem cells into skeletal muscle cells by combining RNA-based MYOD1-expression and POU5F1-silencing
Article Snippet: Paragraph title: RNA-sequencing ... The cDNA libraries of siControl-treated cells, siPOU5F1-treated cells, and siPOU5F1/synMYOD1-treated cells were prepared from 500 ng of each total RNA sample for massive parallel sequencing using an NEBNext Poly(A) mRNA Magnetic Isolation Module and an Ultra Directional RNA Library Prep Kit for Illumina (NEB).

High Throughput Screening Assay:

Article Title: Genome-wide long non-coding RNA screening, identification and characterization in a model microorganism Chlamydomonas reinhardtii
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Article Title: Expression and functional analysis of lncRNAs in the hippocampus of immature rats with status epilepticus, et al. Expression and functional analysis of lncRNAs in the hippocampus of immature rats with status epilepticus
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Synthesized:

Article Title: Genome-wide long non-coding RNA screening, identification and characterization in a model microorganism Chlamydomonas reinhardtii
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Article Title: Chloride intracellular channel 1 (CLIC1) contributes to modulation of cyclic AMP‐activated whole‐cell chloride currents in human bronchial epithelial cells. Chloride intracellular channel 1 (CLIC1) contributes to modulation of cyclic AMP‐activated whole cell chloride currents in human bronchial epithelial cells
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Article Title: Expression and functional analysis of lncRNAs in the hippocampus of immature rats with status epilepticus, et al. Expression and functional analysis of lncRNAs in the hippocampus of immature rats with status epilepticus
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Isolation:

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Article Title: Both modular and single‐domain Type I polyketide synthases are expressed in the brevetoxin‐producing dinoflagellate, Karenia brevis (Dinophyceae)
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Ligation:

Article Title: Chloride intracellular channel 1 (CLIC1) contributes to modulation of cyclic AMP‐activated whole‐cell chloride currents in human bronchial epithelial cells. Chloride intracellular channel 1 (CLIC1) contributes to modulation of cyclic AMP‐activated whole cell chloride currents in human bronchial epithelial cells
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Random Hexamer Labeling:

Article Title: Genome-wide long non-coding RNA screening, identification and characterization in a model microorganism Chlamydomonas reinhardtii
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Article Title: Differential transcription profiles of long non-coding RNAs in primary human brain microvascular endothelial cells in response to meningitic Escherichia coli
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Article Title: Expression and functional analysis of lncRNAs in the hippocampus of immature rats with status epilepticus, et al. Expression and functional analysis of lncRNAs in the hippocampus of immature rats with status epilepticus
Article Snippet: Briefly speaking, equivalent total RNAs were used to construct the sulphur‐replete and sulphur‐deprived libraries by NEB Next® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB) following manufacturer's recommendations. .. RNA was broken into fragments by divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer and converted to first strand cDNA performed with random hexamer primer and M‐Mu LV Reverse Transcriptase.

Construct:

Article Title: Genome-wide long non-coding RNA screening, identification and characterization in a model microorganism Chlamydomonas reinhardtii
Article Snippet: .. LncRNA library construction and high-throughput sequencing Equivalent total RNAs from TAP and TAP-S cultured algal cells were used to construct the sulfur-replete and sulfur-deprived libraries by NEB Next® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations. .. Briefly, RNA was broken into fragments by divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer, and converted to first strand cDNA using random hexamer primer and M-MuLV Reverse Transcriptase.

Article Title: Tissue-Specific Transcriptome Analysis Reveals Multiple Responses to Salt Stress in Populus euphratica Seedlings
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Article Title: Differential transcription profiles of long non-coding RNAs in primary human brain microvascular endothelial cells in response to meningitic Escherichia coli
Article Snippet: .. The library was constructed with the NEB Next® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, Ipswich, MA, USA), following the manufacturer’s recommendations. .. Briefly, fragmentation was carried out using divalent cations at an elevated temperature in NEBNext First-Strand Synthesis Reaction Buffer (5×).

Article Title: Expression and functional analysis of lncRNAs in the hippocampus of immature rats with status epilepticus, et al. Expression and functional analysis of lncRNAs in the hippocampus of immature rats with status epilepticus
Article Snippet: .. Briefly speaking, equivalent total RNAs were used to construct the sulphur‐replete and sulphur‐deprived libraries by NEB Next® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB) following manufacturer's recommendations. .. RNA was broken into fragments by divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer and converted to first strand cDNA performed with random hexamer primer and M‐Mu LV Reverse Transcriptase.

Article Title: Global miRNA, lncRNA, and mRNA Transcriptome Profiling of Endometrial Epithelial Cells Reveals Genes Related to Porcine Reproductive Failure Caused by Porcine Reproductive and Respiratory Syndrome Virus
Article Snippet: The sequencing libraries were generated using rRNA-depleted RNA with a NEB Next® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA). .. The constructed libraries were evaluated on an Agilent Bioanalyzer 2100 system ( ).

Produced:

Article Title: Efficient differentiation of human pluripotent stem cells into skeletal muscle cells by combining RNA-based MYOD1-expression and POU5F1-silencing
Article Snippet: RNA-sequencing The cDNA libraries of siControl-treated cells, siPOU5F1-treated cells, and siPOU5F1/synMYOD1-treated cells were prepared from 500 ng of each total RNA sample for massive parallel sequencing using an NEBNext Poly(A) mRNA Magnetic Isolation Module and an Ultra Directional RNA Library Prep Kit for Illumina (NEB). .. The cDNA library produced ranged from 400 to 1000 bp, including the adaptor sequences.

Article Title: Efficient differentiation of human pluripotent stem cells into skeletal muscle cells by combining RNA-based MYOD1-expression and POU5F1-silencing
Article Snippet: The cDNA libraries of siControl-treated cells, siPOU5F1-treated cells, and siPOU5F1/synMYOD1-treated cells were prepared from 500 ng of each total RNA sample for massive parallel sequencing using an NEBNext Poly(A) mRNA Magnetic Isolation Module and an Ultra Directional RNA Library Prep Kit for Illumina (NEB). .. The cDNA library produced ranged from 400 to 1000 bp, including the adaptor sequences.

Polymerase Chain Reaction:

Article Title: Chloride intracellular channel 1 (CLIC1) contributes to modulation of cyclic AMP‐activated whole‐cell chloride currents in human bronchial epithelial cells. Chloride intracellular channel 1 (CLIC1) contributes to modulation of cyclic AMP‐activated whole cell chloride currents in human bronchial epithelial cells
Article Snippet: A sequencing library was prepared using the Next Ultra Directional RNA Library Prep kit for Illumina (New England Biolabs). mRNA was poly‐A selected using Dynabeads Oligo (dT)25 (Life technologies). cDNA was synthesized using random primers. .. Finally, a cDNA library was prepared by end‐repair, phosphorylation, A‐tailing, adapter ligation, and PCR amplification.

Generated:

Article Title: Tissue-Specific Transcriptome Analysis Reveals Multiple Responses to Salt Stress in Populus euphratica Seedlings
Article Snippet: Whole transcriptome libraries were constructed using the New England Biolabs Next, Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA), in accordance with the manufacturer’s instructions. .. The resulting libraries were initially sequenced on a HiSeq 2500 instrument (Illumina, San Diego, CA, USA) and paired-end, 125 nucleotide reads were generated.

Article Title: Chloride intracellular channel 1 (CLIC1) contributes to modulation of cyclic AMP‐activated whole‐cell chloride currents in human bronchial epithelial cells. Chloride intracellular channel 1 (CLIC1) contributes to modulation of cyclic AMP‐activated whole cell chloride currents in human bronchial epithelial cells
Article Snippet: A sequencing library was prepared using the Next Ultra Directional RNA Library Prep kit for Illumina (New England Biolabs). mRNA was poly‐A selected using Dynabeads Oligo (dT)25 (Life technologies). cDNA was synthesized using random primers. .. Paired‐end sequencing was performed on Illumina NextSeq500 with approximately 40 million reads per sample generated.

Article Title: Inhibition of prostaglandin E2 receptor 4 by lnc000908 to promote the endothelial‐mesenchymal transition participation in cardiac remodelling, et al. Inhibition of prostaglandin E2 receptor 4 by lnc000908 to promote the endothelial‐mesenchymal transition participation in cardiac remodelling
Article Snippet: .. In addition, sequencing libraries were generated using the rRNA‐depleted RNA by NEB Next® Ultra™ Directional RNA Library Prep Kit from Illumina® (NEB). .. After cluster generation, the libraries were sequenced on an Illumina HiSeq 2500 platform, and 125‐bp paired‐end reads were generated.

Article Title: Both modular and single‐domain Type I polyketide synthases are expressed in the brevetoxin‐producing dinoflagellate, Karenia brevis (Dinophyceae)
Article Snippet: .. Nine stranded libraries were generated using the NEBNext Ultra Directional RNA Library Prep Kit (Illumina, Hayward, CA, USA) from total RNA of log phase cultures under control, exponential growth conditions (n = 3), or cultures exposed to 30 min (n = 3) or 60 min (n = 3) of 6°C heat shock prior to harvest as described in Fridey ( ). .. Sequencing of these libraries was performed on an Illumina Hiseq 2500 sequencer, at a depth of ~25 million, 125 nt, single end reads per library.

Article Title: Global miRNA, lncRNA, and mRNA Transcriptome Profiling of Endometrial Epithelial Cells Reveals Genes Related to Porcine Reproductive Failure Caused by Porcine Reproductive and Respiratory Syndrome Virus
Article Snippet: .. The sequencing libraries were generated using rRNA-depleted RNA with a NEB Next® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA). .. The constructed libraries were evaluated on an Agilent Bioanalyzer 2100 system ( ).

cDNA Library Assay:

Article Title: Efficient differentiation of human pluripotent stem cells into skeletal muscle cells by combining RNA-based MYOD1-expression and POU5F1-silencing
Article Snippet: RNA-sequencing The cDNA libraries of siControl-treated cells, siPOU5F1-treated cells, and siPOU5F1/synMYOD1-treated cells were prepared from 500 ng of each total RNA sample for massive parallel sequencing using an NEBNext Poly(A) mRNA Magnetic Isolation Module and an Ultra Directional RNA Library Prep Kit for Illumina (NEB). .. The cDNA library produced ranged from 400 to 1000 bp, including the adaptor sequences.

Article Title: Chloride intracellular channel 1 (CLIC1) contributes to modulation of cyclic AMP‐activated whole‐cell chloride currents in human bronchial epithelial cells. Chloride intracellular channel 1 (CLIC1) contributes to modulation of cyclic AMP‐activated whole cell chloride currents in human bronchial epithelial cells
Article Snippet: A sequencing library was prepared using the Next Ultra Directional RNA Library Prep kit for Illumina (New England Biolabs). mRNA was poly‐A selected using Dynabeads Oligo (dT)25 (Life technologies). cDNA was synthesized using random primers. .. Finally, a cDNA library was prepared by end‐repair, phosphorylation, A‐tailing, adapter ligation, and PCR amplification.

Article Title: Integrated omics data of two annual ryegrass (Lolium multiflorum L.) genotypes reveals core metabolic processes under drought stress
Article Snippet: .. A mass of 5 μg per sample was collected for cDNA library construction, using the NEB Next® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA). .. The cDNA Library was validated by two different methods to determine the average molecular length: 1) the Agilent 2100 Bioanalyzer (Agilent DNA 1000 Reagents; Agilent Technologies) and 2) real-time quantitative PCR (QPCR; TaqMan Probe; Thermo Fisher Scientific, Waltham, MA, USA).

Article Title: Efficient differentiation of human pluripotent stem cells into skeletal muscle cells by combining RNA-based MYOD1-expression and POU5F1-silencing
Article Snippet: The cDNA libraries of siControl-treated cells, siPOU5F1-treated cells, and siPOU5F1/synMYOD1-treated cells were prepared from 500 ng of each total RNA sample for massive parallel sequencing using an NEBNext Poly(A) mRNA Magnetic Isolation Module and an Ultra Directional RNA Library Prep Kit for Illumina (NEB). .. The cDNA library produced ranged from 400 to 1000 bp, including the adaptor sequences.

Cell Culture:

Article Title: Genome-wide long non-coding RNA screening, identification and characterization in a model microorganism Chlamydomonas reinhardtii
Article Snippet: .. LncRNA library construction and high-throughput sequencing Equivalent total RNAs from TAP and TAP-S cultured algal cells were used to construct the sulfur-replete and sulfur-deprived libraries by NEB Next® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations. .. Briefly, RNA was broken into fragments by divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer, and converted to first strand cDNA using random hexamer primer and M-MuLV Reverse Transcriptase.

Ethanol Precipitation:

Article Title: Global miRNA, lncRNA, and mRNA Transcriptome Profiling of Endometrial Epithelial Cells Reveals Genes Related to Porcine Reproductive Failure Caused by Porcine Reproductive and Respiratory Syndrome Virus
Article Snippet: In one sample, ribosomal RNA was removed by Epicenter Ribo-zero™ rRNA Removal Kit (Epicenter, Madison, WI, USA), and residual RNAs were cleaned by ethanol precipitation. .. The sequencing libraries were generated using rRNA-depleted RNA with a NEB Next® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA).

Sequencing:

Article Title: Genome-wide long non-coding RNA screening, identification and characterization in a model microorganism Chlamydomonas reinhardtii
Article Snippet: .. LncRNA library construction and high-throughput sequencing Equivalent total RNAs from TAP and TAP-S cultured algal cells were used to construct the sulfur-replete and sulfur-deprived libraries by NEB Next® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations. .. Briefly, RNA was broken into fragments by divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer, and converted to first strand cDNA using random hexamer primer and M-MuLV Reverse Transcriptase.

Article Title: Tissue-Specific Transcriptome Analysis Reveals Multiple Responses to Salt Stress in Populus euphratica Seedlings
Article Snippet: Paragraph title: 2.2. Illumina Sequencing ... Whole transcriptome libraries were constructed using the New England Biolabs Next, Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA), in accordance with the manufacturer’s instructions.

Article Title: Efficient differentiation of human pluripotent stem cells into skeletal muscle cells by combining RNA-based MYOD1-expression and POU5F1-silencing
Article Snippet: .. RNA-sequencing The cDNA libraries of siControl-treated cells, siPOU5F1-treated cells, and siPOU5F1/synMYOD1-treated cells were prepared from 500 ng of each total RNA sample for massive parallel sequencing using an NEBNext Poly(A) mRNA Magnetic Isolation Module and an Ultra Directional RNA Library Prep Kit for Illumina (NEB). .. The cDNA library produced ranged from 400 to 1000 bp, including the adaptor sequences.

Article Title: Identifying TF-miRNA-mRNA regulatory modules in nitidine chloride treated HCC xenograft of nude mice
Article Snippet: .. The miRNA and mRNA sequencing libraries were established using the NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (NEB, USA) and the rRNA-depleted RNA by NEB Next® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA), respectively, following the manufacturer’s recommendations. .. The library quality was monitored on the Agilent Bioanalyzer 2100 system.

Article Title: Chloride intracellular channel 1 (CLIC1) contributes to modulation of cyclic AMP‐activated whole‐cell chloride currents in human bronchial epithelial cells. Chloride intracellular channel 1 (CLIC1) contributes to modulation of cyclic AMP‐activated whole cell chloride currents in human bronchial epithelial cells
Article Snippet: .. A sequencing library was prepared using the Next Ultra Directional RNA Library Prep kit for Illumina (New England Biolabs). mRNA was poly‐A selected using Dynabeads Oligo (dT)25 (Life technologies). cDNA was synthesized using random primers. .. Finally, a cDNA library was prepared by end‐repair, phosphorylation, A‐tailing, adapter ligation, and PCR amplification.

Article Title: Integrated omics data of two annual ryegrass (Lolium multiflorum L.) genotypes reveals core metabolic processes under drought stress
Article Snippet: Paragraph title: Transcriptome sequencing and analysis ... A mass of 5 μg per sample was collected for cDNA library construction, using the NEB Next® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA).

Article Title: Expression and functional analysis of lncRNAs in the hippocampus of immature rats with status epilepticus, et al. Expression and functional analysis of lncRNAs in the hippocampus of immature rats with status epilepticus
Article Snippet: Paragraph title: LncRNA library construction and high‐throughput sequencing ... Briefly speaking, equivalent total RNAs were used to construct the sulphur‐replete and sulphur‐deprived libraries by NEB Next® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB) following manufacturer's recommendations.

Article Title: Inhibition of prostaglandin E2 receptor 4 by lnc000908 to promote the endothelial‐mesenchymal transition participation in cardiac remodelling, et al. Inhibition of prostaglandin E2 receptor 4 by lnc000908 to promote the endothelial‐mesenchymal transition participation in cardiac remodelling
Article Snippet: .. In addition, sequencing libraries were generated using the rRNA‐depleted RNA by NEB Next® Ultra™ Directional RNA Library Prep Kit from Illumina® (NEB). .. After cluster generation, the libraries were sequenced on an Illumina HiSeq 2500 platform, and 125‐bp paired‐end reads were generated.

Article Title: Both modular and single‐domain Type I polyketide synthases are expressed in the brevetoxin‐producing dinoflagellate, Karenia brevis (Dinophyceae)
Article Snippet: These libraries generated ~25 million 50 nt paired‐end sequencing reads on Illumina HiSeq2000. .. Nine stranded libraries were generated using the NEBNext Ultra Directional RNA Library Prep Kit (Illumina, Hayward, CA, USA) from total RNA of log phase cultures under control, exponential growth conditions (n = 3), or cultures exposed to 30 min (n = 3) or 60 min (n = 3) of 6°C heat shock prior to harvest as described in Fridey ( ).

Article Title: Global miRNA, lncRNA, and mRNA Transcriptome Profiling of Endometrial Epithelial Cells Reveals Genes Related to Porcine Reproductive Failure Caused by Porcine Reproductive and Respiratory Syndrome Virus
Article Snippet: .. The sequencing libraries were generated using rRNA-depleted RNA with a NEB Next® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA). .. The constructed libraries were evaluated on an Agilent Bioanalyzer 2100 system ( ).

Article Title: Efficient differentiation of human pluripotent stem cells into skeletal muscle cells by combining RNA-based MYOD1-expression and POU5F1-silencing
Article Snippet: .. The cDNA libraries of siControl-treated cells, siPOU5F1-treated cells, and siPOU5F1/synMYOD1-treated cells were prepared from 500 ng of each total RNA sample for massive parallel sequencing using an NEBNext Poly(A) mRNA Magnetic Isolation Module and an Ultra Directional RNA Library Prep Kit for Illumina (NEB). .. The cDNA library produced ranged from 400 to 1000 bp, including the adaptor sequences.

Real-time Polymerase Chain Reaction:

Article Title: Tissue-Specific Transcriptome Analysis Reveals Multiple Responses to Salt Stress in Populus euphratica Seedlings
Article Snippet: Whole transcriptome libraries were constructed using the New England Biolabs Next, Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA), in accordance with the manufacturer’s instructions. .. The libraries were controlled for quality and quantified using a BioAnalyzer 2100 system and quantitative PCR (qPCR) (Kapa Biosystems, Woburn, MA, USA).

Article Title: Integrated omics data of two annual ryegrass (Lolium multiflorum L.) genotypes reveals core metabolic processes under drought stress
Article Snippet: A mass of 5 μg per sample was collected for cDNA library construction, using the NEB Next® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA). .. The cDNA Library was validated by two different methods to determine the average molecular length: 1) the Agilent 2100 Bioanalyzer (Agilent DNA 1000 Reagents; Agilent Technologies) and 2) real-time quantitative PCR (QPCR; TaqMan Probe; Thermo Fisher Scientific, Waltham, MA, USA).

Spectrophotometry:

Article Title: Identifying TF-miRNA-mRNA regulatory modules in nitidine chloride treated HCC xenograft of nude mice
Article Snippet: RNA purity was detected using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). .. The miRNA and mRNA sequencing libraries were established using the NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (NEB, USA) and the rRNA-depleted RNA by NEB Next® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA), respectively, following the manufacturer’s recommendations.

RNA Extraction:

Article Title: Chloride intracellular channel 1 (CLIC1) contributes to modulation of cyclic AMP‐activated whole‐cell chloride currents in human bronchial epithelial cells. Chloride intracellular channel 1 (CLIC1) contributes to modulation of cyclic AMP‐activated whole cell chloride currents in human bronchial epithelial cells
Article Snippet: Quantification of CLIC homologs in hBEC by RNA sequencing Total RNA was extracted from hBECs from three donors (Lonza, UK) at either passage 3 or 4 using the GenElute Mammalian Total RNA Miniprep Kit (Sigma‐Aldrich) or the RNeasy RNA extraction kit (Qiagen). .. A sequencing library was prepared using the Next Ultra Directional RNA Library Prep kit for Illumina (New England Biolabs). mRNA was poly‐A selected using Dynabeads Oligo (dT)25 (Life technologies). cDNA was synthesized using random primers.

Flow Cytometry:

Article Title: Integrated omics data of two annual ryegrass (Lolium multiflorum L.) genotypes reveals core metabolic processes under drought stress
Article Snippet: A mass of 5 μg per sample was collected for cDNA library construction, using the NEB Next® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA). .. The qualified libraries (average length of fragment was between 250 and 350 bp) were amplified on the cBot System to generate the cluster on the flow cell using the TruSeq PE Cluster Kit V3-cBot-HS (Illumina, San Diego, CA, USA).

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    Illumina Inc ultra directional rna library prep kit
    POU5F1 expression is stably sustained in <t>MYOD1-mRNA</t> (synMYOD1)-treated hESCs. ( a ) synMYOD1 was synthesized in vitro with T7 <t>RNA</t> polymerase. The template cDNA was flanked by 5′UTR and 3′UTR of alpha-globin with an oligo(T) 120 for adding a polyA tail. ARCA (5′cap analog), pseudo-UTP, and 5-methyl-CTP were incorporated to increase mRNA stability and translation efficiency. ( b ) The percentage of mRNA transfection in hESCs was tested using synthetic mRNA encoding Emerald GFP by FACS analysis. ( c ) Schematic diagram of the transfection protocol. hESCs were transfected with synMYOD1 once on day 0, twice on day 1, and once on day 2. ( d ) Immunostaining analysis for MyHC in the synMYOD1-transfected cells. Nuclei were stained with DAPI. The percentage of MyHC-stained cells is shown (mean ± SEM from four independent biological replicates). Scale bar: 200 μm. ( e ) Immunostaining analysis for POU5F1 in the synMYOD1-transfected cells at day 0 to day 3 post transfection. MYOD1 was detected by a MYOD1 specific antibody. Nuclei were stained with DAPI. Scale bar: 10 μm. ( f ) Immunostaining analysis for NANOG in the synMYOD1-transfected cells at day 0 to day 3 post transfection. MYOD1 was detected by the specific antibody. Nuclei were stained with DAPI. Scale bar: 10 μm. ( g ) qRT-PCR analysis for POU5F1 expression day 0 to day 3 after transfection (mean ± SEM from two independent biological replicates). ( h .
    Ultra Directional Rna Library Prep Kit, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 98/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    POU5F1 expression is stably sustained in MYOD1-mRNA (synMYOD1)-treated hESCs. ( a ) synMYOD1 was synthesized in vitro with T7 RNA polymerase. The template cDNA was flanked by 5′UTR and 3′UTR of alpha-globin with an oligo(T) 120 for adding a polyA tail. ARCA (5′cap analog), pseudo-UTP, and 5-methyl-CTP were incorporated to increase mRNA stability and translation efficiency. ( b ) The percentage of mRNA transfection in hESCs was tested using synthetic mRNA encoding Emerald GFP by FACS analysis. ( c ) Schematic diagram of the transfection protocol. hESCs were transfected with synMYOD1 once on day 0, twice on day 1, and once on day 2. ( d ) Immunostaining analysis for MyHC in the synMYOD1-transfected cells. Nuclei were stained with DAPI. The percentage of MyHC-stained cells is shown (mean ± SEM from four independent biological replicates). Scale bar: 200 μm. ( e ) Immunostaining analysis for POU5F1 in the synMYOD1-transfected cells at day 0 to day 3 post transfection. MYOD1 was detected by a MYOD1 specific antibody. Nuclei were stained with DAPI. Scale bar: 10 μm. ( f ) Immunostaining analysis for NANOG in the synMYOD1-transfected cells at day 0 to day 3 post transfection. MYOD1 was detected by the specific antibody. Nuclei were stained with DAPI. Scale bar: 10 μm. ( g ) qRT-PCR analysis for POU5F1 expression day 0 to day 3 after transfection (mean ± SEM from two independent biological replicates). ( h .

    Journal: Scientific Reports

    Article Title: Efficient differentiation of human pluripotent stem cells into skeletal muscle cells by combining RNA-based MYOD1-expression and POU5F1-silencing

    doi: 10.1038/s41598-017-19114-y

    Figure Lengend Snippet: POU5F1 expression is stably sustained in MYOD1-mRNA (synMYOD1)-treated hESCs. ( a ) synMYOD1 was synthesized in vitro with T7 RNA polymerase. The template cDNA was flanked by 5′UTR and 3′UTR of alpha-globin with an oligo(T) 120 for adding a polyA tail. ARCA (5′cap analog), pseudo-UTP, and 5-methyl-CTP were incorporated to increase mRNA stability and translation efficiency. ( b ) The percentage of mRNA transfection in hESCs was tested using synthetic mRNA encoding Emerald GFP by FACS analysis. ( c ) Schematic diagram of the transfection protocol. hESCs were transfected with synMYOD1 once on day 0, twice on day 1, and once on day 2. ( d ) Immunostaining analysis for MyHC in the synMYOD1-transfected cells. Nuclei were stained with DAPI. The percentage of MyHC-stained cells is shown (mean ± SEM from four independent biological replicates). Scale bar: 200 μm. ( e ) Immunostaining analysis for POU5F1 in the synMYOD1-transfected cells at day 0 to day 3 post transfection. MYOD1 was detected by a MYOD1 specific antibody. Nuclei were stained with DAPI. Scale bar: 10 μm. ( f ) Immunostaining analysis for NANOG in the synMYOD1-transfected cells at day 0 to day 3 post transfection. MYOD1 was detected by the specific antibody. Nuclei were stained with DAPI. Scale bar: 10 μm. ( g ) qRT-PCR analysis for POU5F1 expression day 0 to day 3 after transfection (mean ± SEM from two independent biological replicates). ( h .

    Article Snippet: The cDNA libraries of siControl-treated cells, siPOU5F1-treated cells, and siPOU5F1/synMYOD1-treated cells were prepared from 500 ng of each total RNA sample for massive parallel sequencing using an NEBNext Poly(A) mRNA Magnetic Isolation Module and an Ultra Directional RNA Library Prep Kit for Illumina (NEB).

    Techniques: Expressing, Stable Transfection, Synthesized, In Vitro, Transfection, FACS, Immunostaining, Staining, Quantitative RT-PCR

    Transfection of si RNA specifically targeted against CLIC 1 significantly reduces both mRNA and protein expressions of CLIC 1 in cultured hBEC cells . Forty‐eight hours posttransfection of CLIC 1‐targeted si RNA (1 nmol/L) both mRNA (duplicate n = 3) (A) and protein (B and C) expression of CLIC 1 was reduced in cultured hBEC cells (B is a representative image of n = 6, **** denotes P

    Journal: Physiological Reports

    Article Title: Chloride intracellular channel 1 (CLIC1) contributes to modulation of cyclic AMP‐activated whole‐cell chloride currents in human bronchial epithelial cells. Chloride intracellular channel 1 (CLIC1) contributes to modulation of cyclic AMP‐activated whole cell chloride currents in human bronchial epithelial cells

    doi: 10.14814/phy2.13508

    Figure Lengend Snippet: Transfection of si RNA specifically targeted against CLIC 1 significantly reduces both mRNA and protein expressions of CLIC 1 in cultured hBEC cells . Forty‐eight hours posttransfection of CLIC 1‐targeted si RNA (1 nmol/L) both mRNA (duplicate n = 3) (A) and protein (B and C) expression of CLIC 1 was reduced in cultured hBEC cells (B is a representative image of n = 6, **** denotes P

    Article Snippet: A sequencing library was prepared using the Next Ultra Directional RNA Library Prep kit for Illumina (New England Biolabs). mRNA was poly‐A selected using Dynabeads Oligo (dT)25 (Life technologies). cDNA was synthesized using random primers.

    Techniques: Transfection, Cell Culture, Expressing

    POU5F1 expression is stably sustained in MYOD1-mRNA (synMYOD1)-treated hESCs. ( a ) synMYOD1 was synthesized in vitro with T7 RNA polymerase. The template cDNA was flanked by 5′UTR and 3′UTR of alpha-globin with an oligo(T) 120 for adding a polyA tail. ARCA (5′cap analog), pseudo-UTP, and 5-methyl-CTP were incorporated to increase mRNA stability and translation efficiency. ( b ) The percentage of mRNA transfection in hESCs was tested using synthetic mRNA encoding Emerald GFP by FACS analysis. ( c ) Schematic diagram of the transfection protocol. hESCs were transfected with synMYOD1 once on day 0, twice on day 1, and once on day 2. ( d ) Immunostaining analysis for MyHC in the synMYOD1-transfected cells. Nuclei were stained with DAPI. The percentage of MyHC-stained cells is shown (mean ± SEM from four independent biological replicates). Scale bar: 200 μm. ( e ) Immunostaining analysis for POU5F1 in the synMYOD1-transfected cells at day 0 to day 3 post transfection. MYOD1 was detected by a MYOD1 specific antibody. Nuclei were stained with DAPI. Scale bar: 10 μm. ( f ) Immunostaining analysis for NANOG in the synMYOD1-transfected cells at day 0 to day 3 post transfection. MYOD1 was detected by the specific antibody. Nuclei were stained with DAPI. Scale bar: 10 μm. ( g ) qRT-PCR analysis for POU5F1 expression day 0 to day 3 after transfection (mean ± SEM from two independent biological replicates). ( h ) Immunoblotting analysis for POU5F1 in the synMYOD1-transfected cells at day 3 post transfection. MYOD1 was detected by the specific antibody. The H3 antibody was used as a loading control. The relative intensities of POU5F1 signals normalized by H3 were compared between no transfection and synMYOD1 transfection (mean ± SEM from three independent biological replicates). NS: not significant. Uncropped images of the blots for Fig. 1h are shown in Supplementary Figure 7 .

    Journal: Scientific Reports

    Article Title: Efficient differentiation of human pluripotent stem cells into skeletal muscle cells by combining RNA-based MYOD1-expression and POU5F1-silencing

    doi: 10.1038/s41598-017-19114-y

    Figure Lengend Snippet: POU5F1 expression is stably sustained in MYOD1-mRNA (synMYOD1)-treated hESCs. ( a ) synMYOD1 was synthesized in vitro with T7 RNA polymerase. The template cDNA was flanked by 5′UTR and 3′UTR of alpha-globin with an oligo(T) 120 for adding a polyA tail. ARCA (5′cap analog), pseudo-UTP, and 5-methyl-CTP were incorporated to increase mRNA stability and translation efficiency. ( b ) The percentage of mRNA transfection in hESCs was tested using synthetic mRNA encoding Emerald GFP by FACS analysis. ( c ) Schematic diagram of the transfection protocol. hESCs were transfected with synMYOD1 once on day 0, twice on day 1, and once on day 2. ( d ) Immunostaining analysis for MyHC in the synMYOD1-transfected cells. Nuclei were stained with DAPI. The percentage of MyHC-stained cells is shown (mean ± SEM from four independent biological replicates). Scale bar: 200 μm. ( e ) Immunostaining analysis for POU5F1 in the synMYOD1-transfected cells at day 0 to day 3 post transfection. MYOD1 was detected by a MYOD1 specific antibody. Nuclei were stained with DAPI. Scale bar: 10 μm. ( f ) Immunostaining analysis for NANOG in the synMYOD1-transfected cells at day 0 to day 3 post transfection. MYOD1 was detected by the specific antibody. Nuclei were stained with DAPI. Scale bar: 10 μm. ( g ) qRT-PCR analysis for POU5F1 expression day 0 to day 3 after transfection (mean ± SEM from two independent biological replicates). ( h ) Immunoblotting analysis for POU5F1 in the synMYOD1-transfected cells at day 3 post transfection. MYOD1 was detected by the specific antibody. The H3 antibody was used as a loading control. The relative intensities of POU5F1 signals normalized by H3 were compared between no transfection and synMYOD1 transfection (mean ± SEM from three independent biological replicates). NS: not significant. Uncropped images of the blots for Fig. 1h are shown in Supplementary Figure 7 .

    Article Snippet: RNA-sequencing The cDNA libraries of siControl-treated cells, siPOU5F1-treated cells, and siPOU5F1/synMYOD1-treated cells were prepared from 500 ng of each total RNA sample for massive parallel sequencing using an NEBNext Poly(A) mRNA Magnetic Isolation Module and an Ultra Directional RNA Library Prep Kit for Illumina (NEB).

    Techniques: Expressing, Stable Transfection, Synthesized, In Vitro, Transfection, FACS, Immunostaining, Staining, Quantitative RT-PCR