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Mrna Library Preparation Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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quality analysis illumina truseq stranded mrna library preparation kit (Thermo Fisher)

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Quality Analysis Illumina Truseq Stranded Mrna Library Preparation Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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collibri 3 mrna library preparation kit (Thermo Fisher)

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Collibri 3 Mrna Library Preparation Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Mrna Seq Library Preparation, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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$(A) Microscopic images of JM8 wild-type mouse embryonic stem cells (mESCs) and in vitro–derived mouse neural stem cell (mNSC) cultures. Scale bar is 100 μm. (B) RT-qPCR analysis of pluripotency-associated genes ( Nanog , Oct4 , and Rex1 ) and neural stem cell gene markers ( Fabp7 , Slc1a3 , and Nestin ) in mESCs and mNSCs. Expression is normalized to Gapdh and RNA18s . (C) Immunofluorescence images showing the expression of pluripotency (Oct4, green) and NSC (Nestin, green) protein markers in mESC and mNSC cultures. DAPI stains nuclear DNA (blue). Scale bar is 10 μm. (D) Expression of Bmal1 in mESCs and NSCs measured by RT-qPCR. Expression of Hmbs was included as a control of a transcriptionally active and functional gene. Expression was normalized to Gapdh and RNA18s and multiplied by 10 to facilitate representation. (E) Western blot analysis of whole-cell extracts comparing Bmal1 protein levels in mESCs and mNSCs. B-actin provides a loading control. (F) RT-qPCR analysis comparing expression of Bmal1 in mESCs grown in FBS + LIF <t>or</t> <t>2i</t> + LIF media. Expression was normalized to Gapdh and RNA18s and multiplied by 10 to facilitate representation. (G) Western blot analysis of whole-cell extracts comparing Bmal1 protein levels in mESCs grown in FBS + LIF and 2i + LIF media. B-actin provides a loading control. (H) Single-cell <t>mRNA</t> expression values of Bmal1 during indicated stages of preimplantation development calculated using previously published datasets . (I) Analysis of Bmal1 expression in Oct4 low and high individual cells during the blastocyst stage (early, mid, and late). Mean and SEM is shown. (J) Western blot analysis of Bmal1 protein localization in cell fractionation analysis of mESCs and mNSCs. Cytoplasmic (C) and nuclear (N) fractions are indicated. Ezh2 was used as a nuclear control. (B, D, F) Mean and SEM of three independent experiments are shown in (B, D, F). (B, I) Asterisks (*) in (B, I) indicate significant difference (t-Student P < 0.05).$
Mrna Library Preparation, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mrna library preparation - by Bioz Stars, 2023-01

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1) Product Images from "The molecular clock protein Bmal1 regulates cell differentiation in mouse embryonic stem cells"

Article Title: The molecular clock protein Bmal1 regulates cell differentiation in mouse embryonic stem cells

Journal: Life Science Alliance

doi: 10.26508/lsa.201900535

$(A) Microscopic images of JM8 wild-type mouse embryonic stem cells (mESCs) and in vitro–derived mouse neural stem cell (mNSC) cultures. Scale bar is 100 μm. (B) RT-qPCR analysis of pluripotency-associated genes ( Nanog , Oct4 , and Rex1 ) and neural stem cell gene markers ( Fabp7 , Slc1a3 , and Nestin ) in mESCs and mNSCs. Expression is normalized to Gapdh and RNA18s . (C) Immunofluorescence images showing the expression of pluripotency (Oct4, green) and NSC (Nestin, green) protein markers in mESC and mNSC cultures. DAPI stains nuclear DNA (blue). Scale bar is 10 μm. (D) Expression of Bmal1 in mESCs and NSCs measured by RT-qPCR. Expression of Hmbs was included as a control of a transcriptionally active and functional gene. Expression was normalized to Gapdh and RNA18s and multiplied by 10 to facilitate representation. (E) Western blot analysis of whole-cell extracts comparing Bmal1 protein levels in mESCs and mNSCs. B-actin provides a loading control. (F) RT-qPCR analysis comparing expression of Bmal1 in mESCs grown in FBS + LIF or 2i + LIF media. Expression was normalized to Gapdh and RNA18s and multiplied by 10 to facilitate representation. (G) Western blot analysis of whole-cell extracts comparing Bmal1 protein levels in mESCs grown in FBS + LIF and 2i + LIF media. B-actin provides a loading control. (H) Single-cell mRNA expression values of Bmal1 during indicated stages of preimplantation development calculated using previously published datasets . (I) Analysis of Bmal1 expression in Oct4 low and high individual cells during the blastocyst stage (early, mid, and late). Mean and SEM is shown. (J) Western blot analysis of Bmal1 protein localization in cell fractionation analysis of mESCs and mNSCs. Cytoplasmic (C) and nuclear (N) fractions are indicated. Ezh2 was used as a nuclear control. (B, D, F) Mean and SEM of three independent experiments are shown in (B, D, F). (B, I) Asterisks (*) in (B, I) indicate significant difference (t-Student P < 0.05).$
Figure Legend Snippet: (A) Microscopic images of JM8 wild-type mouse embryonic stem cells (mESCs) and in vitro–derived mouse neural stem cell (mNSC) cultures. Scale bar is 100 μm. (B) RT-qPCR analysis of pluripotency-associated genes ( Nanog , Oct4 , and Rex1 ) and neural stem cell gene markers ( Fabp7 , Slc1a3 , and Nestin ) in mESCs and mNSCs. Expression is normalized to Gapdh and RNA18s . (C) Immunofluorescence images showing the expression of pluripotency (Oct4, green) and NSC (Nestin, green) protein markers in mESC and mNSC cultures. DAPI stains nuclear DNA (blue). Scale bar is 10 μm. (D) Expression of Bmal1 in mESCs and NSCs measured by RT-qPCR. Expression of Hmbs was included as a control of a transcriptionally active and functional gene. Expression was normalized to Gapdh and RNA18s and multiplied by 10 to facilitate representation. (E) Western blot analysis of whole-cell extracts comparing Bmal1 protein levels in mESCs and mNSCs. B-actin provides a loading control. (F) RT-qPCR analysis comparing expression of Bmal1 in mESCs grown in FBS + LIF or 2i + LIF media. Expression was normalized to Gapdh and RNA18s and multiplied by 10 to facilitate representation. (G) Western blot analysis of whole-cell extracts comparing Bmal1 protein levels in mESCs grown in FBS + LIF and 2i + LIF media. B-actin provides a loading control. (H) Single-cell mRNA expression values of Bmal1 during indicated stages of preimplantation development calculated using previously published datasets . (I) Analysis of Bmal1 expression in Oct4 low and high individual cells during the blastocyst stage (early, mid, and late). Mean and SEM is shown. (J) Western blot analysis of Bmal1 protein localization in cell fractionation analysis of mESCs and mNSCs. Cytoplasmic (C) and nuclear (N) fractions are indicated. Ezh2 was used as a nuclear control. (B, D, F) Mean and SEM of three independent experiments are shown in (B, D, F). (B, I) Asterisks (*) in (B, I) indicate significant difference (t-Student P < 0.05).

Techniques Used: In Vitro, Derivative Assay, Quantitative RT-PCR, Expressing, Immunofluorescence, Functional Assay, Western Blot, Cell Fractionation

(A) Scheme of the Bmal1 gene (black bars, exons) and the CRISPR/Cas9 strategy used. DNA sequence targeted by the guide RNA (red) and the localization of the PAM domain (blue) are indicated. (B) Alignment of DNA sequences of the Bmal1 gene upon genotyping of Bmal1 −/− mESCs obtained using CRISPR/Cas9. DNA regions targeted by the guide RNA are indicated in red. (C) Microscopy images of Bmal1 −/− and wild-type mESCs grown in the 2i + LIF medium. Scale bar is 100 μm. (D) RT-qPCR analysis of Bmal1 mRNA in wild-type and Bmal1 −/− mESCs. ActB was used as a housekeeping control. Expression was normalized to Gapdh and RNA18s and multiplied by 10 for better representation. n.d. indicates non-detection of transcripts. Mean and SEM of three independent experiments is shown. (E) Western blot analysis of Bmal1 protein using whole-cell extracts of Bmal1 −/− and wild-type mESCs. Lamin B provides a loading control. (F) mRNA expression analysis of Nanog , Oct4 , and Rex1 in Bmal1 −/− and wild-type mESCs by RT-qPCR. Expression was normalized to Gapdh and RNA18s and multiplied by 10 for better representation. Asterisk (*) indicates significant difference (T-student P < 0.05). (G) Western blot analysis of whole-cell extracts comparing the protein levels of Nanog and Oct4 in Bmal1 −/− and wild-type mESCs. Lamin B was used as a loading control. (H) Immunofluorescence analysis of Nanog protein (green) expression in Bmal1 −/− and wild-type mESCs. DAPI stains nuclear DNA (blue). Scale bar is 10 μm. (I) Histogram showing the percentage of cells with low or high intensity of Nanog staining in parental and Bmal1 −/− mESCs. Mean and SEM of three independent experiments is shown. Asterisks (*) indicate significant difference (Fisher’s test, P < 0.01).

Figure Legend Snippet: (A) Scheme of the Bmal1 gene (black bars, exons) and the CRISPR/Cas9 strategy used. DNA sequence targeted by the guide RNA (red) and the localization of the PAM domain (blue) are indicated. (B) Alignment of DNA sequences of the Bmal1 gene upon genotyping of Bmal1 −/− mESCs obtained using CRISPR/Cas9. DNA regions targeted by the guide RNA are indicated in red. (C) Microscopy images of Bmal1 −/− and wild-type mESCs grown in the 2i + LIF medium. Scale bar is 100 μm. (D) RT-qPCR analysis of Bmal1 mRNA in wild-type and Bmal1 −/− mESCs. ActB was used as a housekeeping control. Expression was normalized to Gapdh and RNA18s and multiplied by 10 for better representation. n.d. indicates non-detection of transcripts. Mean and SEM of three independent experiments is shown. (E) Western blot analysis of Bmal1 protein using whole-cell extracts of Bmal1 −/− and wild-type mESCs. Lamin B provides a loading control. (F) mRNA expression analysis of Nanog , Oct4 , and Rex1 in Bmal1 −/− and wild-type mESCs by RT-qPCR. Expression was normalized to Gapdh and RNA18s and multiplied by 10 for better representation. Asterisk (*) indicates significant difference (T-student P < 0.05). (G) Western blot analysis of whole-cell extracts comparing the protein levels of Nanog and Oct4 in Bmal1 −/− and wild-type mESCs. Lamin B was used as a loading control. (H) Immunofluorescence analysis of Nanog protein (green) expression in Bmal1 −/− and wild-type mESCs. DAPI stains nuclear DNA (blue). Scale bar is 10 μm. (I) Histogram showing the percentage of cells with low or high intensity of Nanog staining in parental and Bmal1 −/− mESCs. Mean and SEM of three independent experiments is shown. Asterisks (*) indicate significant difference (Fisher’s test, P < 0.01).

Techniques Used: CRISPR, Sequencing, Microscopy, Quantitative RT-PCR, Expressing, Western Blot, Immunofluorescence, Staining

(A) Gene expression analysis by mRNA-seq of Bmal1 −/− and parental wild-type mESCs grown in the 2i + LIF medium using heat map clustering. Red and blue indicates higher and lower expression, respectively. Biological duplicates (R1 and R2) were carried out for indicated genotypes. (B) MA plot analysis of deregulated genes in Bmal1 −/− mESCs. y-axis indicates fold change expression between Bmal1 −/− and parental mESCs, whereas x-axis shows the mean expression level. Each dot represents one gene. Significantly mis-regulated genes are plotted in red. (C, D, E) Gene enrichment analyses of mis-regulated genes (854 genes with FC > 2 and P < 0.05) in Bmal1 −/− mESCs. Categories are sorted by P -value and the number of genes included in each term are indicated in the x-axis. (F) Comparative expression analysis in Bmal1 −/− and wild-type mESCs of genes involved in pluripotency, PI3K, and MAPK signalling pathways using heat map clustering. (G) Representative images of haematoxylin and eosin staining of teratomas derived from wild-type and Bmal1 −/− mESCs. Tissues corresponding to three germ layers are shown (black arrow): skin for ectoderm, glandular tissue for endoderm, and smooth muscle for mesoderm. Scale bar is 100 μm. (H) Representative images of immunohistochemistry analysis of teratomas derived from wild-type and Bmal1 −/− mESCs. Antibodies against Glial fibrillary acidic protein (ectoderm marker), cytokeratin (endoderm marker), and smooth muscle actin (mesoderm marker) were used. Scale bar is 500 μm. (H, I) Histogram showing quantification of the area of the images positive for indicated antibodies in teratoma immunohistochemistry analysis in (H). Mean and SEM of the analysis of 10 images are shown. Asterisk (*) indicates significant difference (t-Student < 0.05).

Figure Legend Snippet: (A) Gene expression analysis by mRNA-seq of Bmal1 −/− and parental wild-type mESCs grown in the 2i + LIF medium using heat map clustering. Red and blue indicates higher and lower expression, respectively. Biological duplicates (R1 and R2) were carried out for indicated genotypes. (B) MA plot analysis of deregulated genes in Bmal1 −/− mESCs. y-axis indicates fold change expression between Bmal1 −/− and parental mESCs, whereas x-axis shows the mean expression level. Each dot represents one gene. Significantly mis-regulated genes are plotted in red. (C, D, E) Gene enrichment analyses of mis-regulated genes (854 genes with FC > 2 and P < 0.05) in Bmal1 −/− mESCs. Categories are sorted by P -value and the number of genes included in each term are indicated in the x-axis. (F) Comparative expression analysis in Bmal1 −/− and wild-type mESCs of genes involved in pluripotency, PI3K, and MAPK signalling pathways using heat map clustering. (G) Representative images of haematoxylin and eosin staining of teratomas derived from wild-type and Bmal1 −/− mESCs. Tissues corresponding to three germ layers are shown (black arrow): skin for ectoderm, glandular tissue for endoderm, and smooth muscle for mesoderm. Scale bar is 100 μm. (H) Representative images of immunohistochemistry analysis of teratomas derived from wild-type and Bmal1 −/− mESCs. Antibodies against Glial fibrillary acidic protein (ectoderm marker), cytokeratin (endoderm marker), and smooth muscle actin (mesoderm marker) were used. Scale bar is 500 μm. (H, I) Histogram showing quantification of the area of the images positive for indicated antibodies in teratoma immunohistochemistry analysis in (H). Mean and SEM of the analysis of 10 images are shown. Asterisk (*) indicates significant difference (t-Student < 0.05).

Techniques Used: Expressing, Staining, Derivative Assay, Immunohistochemistry, Marker

(A) Table displaying the tendency and number of genes deregulated in Bmal1−/− mESCs annotated in indicated categories. (B) Comparative expression analysis by mRNA-seq in Bmal1−/− and wild-type mESCs of genes involved in Wnt, Ras, and axon guidance signalling pathways using heat map clustering. (C) Western blot analysis of whole-cell extracts comparing total Erk1/2 protein levels in mESC wild-type and Bmal1−/− grown in FBS + LIF and 2i + LIF media. Lamin B provides a loading control. (D) mRNA expression as measured by mRNA-seq in naïve 2i JM8 wild-type (black bars) and Bmal1−/− (grey bars) mESCs of genes previously reported to be induced, repressed, or independent of Erk1/2 signalling in mESCs. (E) mRNA expression as measured by RT-qPCR in primed serum JM8 wild-type (black bars) and Bmal1−/− (grey bars) mESCs of genes previously reported to be induced, repressed, or independent of Erk1/2 signalling in mESCs. (F) mRNA expression as measured by mRNA-seq of representative lineage specifier genes in naïve 2i JM8 wild-type (black bars) and Bmal1−/− (grey bars) mESCs. Expression was normalized to Hmbs and Ywhaz . (G) mRNA expression as measured by RT-qPCR of representative lineage specifier genes in undifferentiated primed serum JM8 wild-type (black bars) and Bmal1−/− (grey bars) mESCs. Expression was normalized to Hmbs and Ywhaz . (E, G) Mean and SEM of three independent experiments are shown in (E, G).

Figure Legend Snippet: (A) Table displaying the tendency and number of genes deregulated in Bmal1−/− mESCs annotated in indicated categories. (B) Comparative expression analysis by mRNA-seq in Bmal1−/− and wild-type mESCs of genes involved in Wnt, Ras, and axon guidance signalling pathways using heat map clustering. (C) Western blot analysis of whole-cell extracts comparing total Erk1/2 protein levels in mESC wild-type and Bmal1−/− grown in FBS + LIF and 2i + LIF media. Lamin B provides a loading control. (D) mRNA expression as measured by mRNA-seq in naïve 2i JM8 wild-type (black bars) and Bmal1−/− (grey bars) mESCs of genes previously reported to be induced, repressed, or independent of Erk1/2 signalling in mESCs. (E) mRNA expression as measured by RT-qPCR in primed serum JM8 wild-type (black bars) and Bmal1−/− (grey bars) mESCs of genes previously reported to be induced, repressed, or independent of Erk1/2 signalling in mESCs. (F) mRNA expression as measured by mRNA-seq of representative lineage specifier genes in naïve 2i JM8 wild-type (black bars) and Bmal1−/− (grey bars) mESCs. Expression was normalized to Hmbs and Ywhaz . (G) mRNA expression as measured by RT-qPCR of representative lineage specifier genes in undifferentiated primed serum JM8 wild-type (black bars) and Bmal1−/− (grey bars) mESCs. Expression was normalized to Hmbs and Ywhaz . (E, G) Mean and SEM of three independent experiments are shown in (E, G).

Techniques Used: Expressing, Western Blot, Quantitative RT-PCR

(A) Alignment of DNA sequences of the Bmal1 gene upon genotyping of Bmal1 −/− (clone #G10) mESCs obtained using CRISPR/Cas9. DNA regions targeted by the guide RNA are indicated in red. (B) Microscopy images of wild-type Bmal1−/− (clone #G10) and JM8 wild-type mESCs grown in the 2i + LIF medium. Scale bar is 100 μm. (C) Microscopic images of gastruloid formation at indicated time points in Bmal1−/− (clone #G10) and JM8 wild-type mESCs. Scale bar is 100 μm. Histogram shows the average diameter of wild-type and Bmal1−/− (clone #G10) gastruloids on day five. (D) mRNA expression of pluripotency (black)-, ectoderm (blue)-, endoderm (green)-, and mesoderm (red)-specific genes at indicated days during gastruloid differentiation as measured by RT-qPCR in JM8 wild-type and Bmal1 −/− (clone #G10) (grey) mESCs. Expression was normalized to Hmbs and Ywhaz . Fold change relative to undifferentiated cells at time zero is represented. Mean and SEM of three independent experiments are shown. Asterisks (*) indicate significant difference (T-student P < 0.05).

Figure Legend Snippet: (A) Alignment of DNA sequences of the Bmal1 gene upon genotyping of Bmal1 −/− (clone #G10) mESCs obtained using CRISPR/Cas9. DNA regions targeted by the guide RNA are indicated in red. (B) Microscopy images of wild-type Bmal1−/− (clone #G10) and JM8 wild-type mESCs grown in the 2i + LIF medium. Scale bar is 100 μm. (C) Microscopic images of gastruloid formation at indicated time points in Bmal1−/− (clone #G10) and JM8 wild-type mESCs. Scale bar is 100 μm. Histogram shows the average diameter of wild-type and Bmal1−/− (clone #G10) gastruloids on day five. (D) mRNA expression of pluripotency (black)-, ectoderm (blue)-, endoderm (green)-, and mesoderm (red)-specific genes at indicated days during gastruloid differentiation as measured by RT-qPCR in JM8 wild-type and Bmal1 −/− (clone #G10) (grey) mESCs. Expression was normalized to Hmbs and Ywhaz . Fold change relative to undifferentiated cells at time zero is represented. Mean and SEM of three independent experiments are shown. Asterisks (*) indicate significant difference (T-student P < 0.05).

Techniques Used: CRISPR, Microscopy, Expressing, Quantitative RT-PCR

mrna library preparation kit (Thermo Fisher)

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mrna library preparation (Thermo Fisher)

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mrna seq library preparation (Thermo Fisher)

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mrna library preparation (Thermo Fisher)

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1) Product Images from "The molecular clock protein Bmal1 regulates cell differentiation in mouse embryonic stem cells"

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