nebnext ultra ii dna library prep  (New England Biolabs)


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    New England Biolabs nebnext ultra ii dna library prep
    Nebnext Ultra Ii Dna Library Prep, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    nebnext ultra ii dna library prep  (New England Biolabs)


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    New England Biolabs nebnext ultra ii dna library prep
    Nebnext Ultra Ii Dna Library Prep, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    nebnext ultra ii dna library prep  (New England Biolabs)


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    New England Biolabs nebnext ultra ii dna library prep
    Nebnext Ultra Ii Dna Library Prep, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    nebnext ultra ii dna library preparation kit  (New England Biolabs)


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    New England Biolabs nebnext ultra ii dna library preparation kit
    KEY RESOURCES TABLE
    Nebnext Ultra Ii Dna Library Preparation Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Transcription factor NFYa controls cardiomyocyte metabolism and proliferation during mouse fetal heart development"

    Article Title: Transcription factor NFYa controls cardiomyocyte metabolism and proliferation during mouse fetal heart development

    Journal: Developmental cell

    doi: 10.1016/j.devcel.2023.10.012

    KEY RESOURCES TABLE
    Figure Legend Snippet: KEY RESOURCES TABLE

    Techniques Used: Produced, Western Blot, Virus, Recombinant, Modification, Protease Inhibitor, Expressing, Software

    nebnext ultra ii dna library prep kit for illumina  (New England Biolabs)


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    New England Biolabs nebnext ultra ii dna library prep kit for illumina
    Nebnext Ultra Ii Dna Library Prep Kit For Illumina, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ultra ii dna library prep kit  (New England Biolabs)


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    New England Biolabs ultra ii dna library prep kit
    a, CasID identified ERV-bound proteins in HEK293 cells, with hits ranked by the fold change of normalized spectral abundance factor (NSAF) relative to control. Label font colors: green, zinc finger proteins; blue, histone and <t>DNA-methylation-related</t> factors; yellow, histone chaperones; purple, N6-methyladenosine-related factors; red, TNRC18. b,c, Scatter plots showing TE families exhibiting expression changes based on RNA-seq of HEK293 cells with endogenous TNRC18 KD (using TNRC18 shRNA (shTNRC18); b) relative to TNRC18 KD followed by TNRC18 re-expression (shTNRC18-rescue; n = 2 independent experiments), or cells with endogenous TNRC18 KO (TNRC18-KO; c) relative to WT (n = 3 independent experiments). The <t>significance</t> <t>cut-off</t> is the fold change in expression over 1.50 and adjusted P value less than 0.01 for transcripts with base mean read counts over 10. Adjusted P value was calculated using negative binomial model-based methods (DESeq2). d, RT–qPCR for TEs (top) and immunity-related genes (bottom) in HEK293 cells with shTNRC18 versus control shRNA (shControl) or shTNRC18- rescue, or TNRC18 KO versus WT (n = 3 independent experiments; plotted as mean ± s.d. after normalization to GAPDH and control samples). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-sided t-test. Exact P values are shown in Supplementary Table 8. e,f, GSEA revealed pathway enrichment in cells with shTNRC18 versus shControl (e), or TNRC18 KO versus WT (f). Immunity-related gene sets are labelled in red. The y axis and x axis show normalized enrichment score (NES) and false discovery rate (FDR) q values, respectively. g, RT–qPCR for TEs in the indicated TNRC18 KO cells versus WT cells (n = 3 independent experiments; plotted as the mean ± s.d. after normalization to GAPDH and to WT). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-sided t-test. Exact P values are shown in Supplementary Table 8. h,i, RNA-seq revealed TEs exhibiting expression changes in SNU-1 cells (h) and HT-29 cells (i) with TNRC18 KO versus WT (n = 3 independent experiments). Significance cut-off is the same as in b,c. Adjusted P values were calculated using negative binomial model-based methods (DESeq2).
    Ultra Ii Dna Library Prep Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "TNRC18 engages H3K9me3 to mediate silencing of endogenous retrotransposons"

    Article Title: TNRC18 engages H3K9me3 to mediate silencing of endogenous retrotransposons

    Journal: Nature

    doi: 10.1038/s41586-023-06688-z

    a, CasID identified ERV-bound proteins in HEK293 cells, with hits ranked by the fold change of normalized spectral abundance factor (NSAF) relative to control. Label font colors: green, zinc finger proteins; blue, histone and DNA-methylation-related factors; yellow, histone chaperones; purple, N6-methyladenosine-related factors; red, TNRC18. b,c, Scatter plots showing TE families exhibiting expression changes based on RNA-seq of HEK293 cells with endogenous TNRC18 KD (using TNRC18 shRNA (shTNRC18); b) relative to TNRC18 KD followed by TNRC18 re-expression (shTNRC18-rescue; n = 2 independent experiments), or cells with endogenous TNRC18 KO (TNRC18-KO; c) relative to WT (n = 3 independent experiments). The significance cut-off is the fold change in expression over 1.50 and adjusted P value less than 0.01 for transcripts with base mean read counts over 10. Adjusted P value was calculated using negative binomial model-based methods (DESeq2). d, RT–qPCR for TEs (top) and immunity-related genes (bottom) in HEK293 cells with shTNRC18 versus control shRNA (shControl) or shTNRC18- rescue, or TNRC18 KO versus WT (n = 3 independent experiments; plotted as mean ± s.d. after normalization to GAPDH and control samples). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-sided t-test. Exact P values are shown in Supplementary Table 8. e,f, GSEA revealed pathway enrichment in cells with shTNRC18 versus shControl (e), or TNRC18 KO versus WT (f). Immunity-related gene sets are labelled in red. The y axis and x axis show normalized enrichment score (NES) and false discovery rate (FDR) q values, respectively. g, RT–qPCR for TEs in the indicated TNRC18 KO cells versus WT cells (n = 3 independent experiments; plotted as the mean ± s.d. after normalization to GAPDH and to WT). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-sided t-test. Exact P values are shown in Supplementary Table 8. h,i, RNA-seq revealed TEs exhibiting expression changes in SNU-1 cells (h) and HT-29 cells (i) with TNRC18 KO versus WT (n = 3 independent experiments). Significance cut-off is the same as in b,c. Adjusted P values were calculated using negative binomial model-based methods (DESeq2).
    Figure Legend Snippet: a, CasID identified ERV-bound proteins in HEK293 cells, with hits ranked by the fold change of normalized spectral abundance factor (NSAF) relative to control. Label font colors: green, zinc finger proteins; blue, histone and DNA-methylation-related factors; yellow, histone chaperones; purple, N6-methyladenosine-related factors; red, TNRC18. b,c, Scatter plots showing TE families exhibiting expression changes based on RNA-seq of HEK293 cells with endogenous TNRC18 KD (using TNRC18 shRNA (shTNRC18); b) relative to TNRC18 KD followed by TNRC18 re-expression (shTNRC18-rescue; n = 2 independent experiments), or cells with endogenous TNRC18 KO (TNRC18-KO; c) relative to WT (n = 3 independent experiments). The significance cut-off is the fold change in expression over 1.50 and adjusted P value less than 0.01 for transcripts with base mean read counts over 10. Adjusted P value was calculated using negative binomial model-based methods (DESeq2). d, RT–qPCR for TEs (top) and immunity-related genes (bottom) in HEK293 cells with shTNRC18 versus control shRNA (shControl) or shTNRC18- rescue, or TNRC18 KO versus WT (n = 3 independent experiments; plotted as mean ± s.d. after normalization to GAPDH and control samples). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-sided t-test. Exact P values are shown in Supplementary Table 8. e,f, GSEA revealed pathway enrichment in cells with shTNRC18 versus shControl (e), or TNRC18 KO versus WT (f). Immunity-related gene sets are labelled in red. The y axis and x axis show normalized enrichment score (NES) and false discovery rate (FDR) q values, respectively. g, RT–qPCR for TEs in the indicated TNRC18 KO cells versus WT cells (n = 3 independent experiments; plotted as the mean ± s.d. after normalization to GAPDH and to WT). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-sided t-test. Exact P values are shown in Supplementary Table 8. h,i, RNA-seq revealed TEs exhibiting expression changes in SNU-1 cells (h) and HT-29 cells (i) with TNRC18 KO versus WT (n = 3 independent experiments). Significance cut-off is the same as in b,c. Adjusted P values were calculated using negative binomial model-based methods (DESeq2).

    Techniques Used: DNA Methylation Assay, Expressing, RNA Sequencing Assay, shRNA, Quantitative RT-PCR

    a. Sanger sequencing to verify the CRISPR-Cas9-induced frameshift and KO of SETDB1 in HEK293 cells. b. Left: Western blot of SETDB1, TNRC18 and H3K9me3 in WT and SETDB1-KO HEK293 cells. Right: Western blot of SETDB1 in HEK293 cells with WT TNRC18, TNRC18 KO and TNRC18 W2858A mutation. Vinculin and Tubulin are the sample processing control. The representative results from 3 independent experiments are shown. c. Heatmap of TNRC18 CUT&RUN signal across ±5 kb at the TNRC18 peaks region in WT and SETDB1-KO HEK293 cells. d. Immunoblotting for H3K9me3, histone H3 acetylation (H3ac) and total H3 in HEK293 cells, either WT or with the homozygous mutation of TNRC18W2858A. Data shown are the representative results from 3 independent experiments. e. The heatmap of CUT&RUN signals of H3ac (left) and H3K9me3 (right) in HEK293 cells, either WT or with the homozygous mutation of TNRC18W2858A, across ±5 kb from the called peaks. f. Box plot showing the log2 values of normalized CUT&RUN sequencing counts in HEK293 cells with homozygous TNRC18W2858A mutation, compared to WT, at the indicated different TE classes. The boundaries of box plots indicate the 25th and 75th percentiles, the center line indicates the median, and the whiskers indicate 1.5× the interquartile range. Sample size of each box plot is listed in Supplementary Table 8. g. Global methylation levels of WT and TNRC18W2858A-mutated HEK293 cells. h. DNA methylation levels of WT and TNRC18W2858A-mutated HEK293 cells at SINE, LTR7Y, LTR12C and CpG_ island. The boundaries of box plots indicate the 25th and 75th percentiles, the center line indicates the median, and the whiskers indicate 1.5× the interquartile range. Sample size of each box plot is listed in Supplementary Table 8. i. The dual-luciferase reporter-based system for assaying the activity of full-length TNRC18 fused to the GAL4’s DNA binding domain (GAL4-DBD), relative to GAL4-DBD alone (n = 6 biologically independent samples; data presented as the mean ±s.d.; ****P < 0.0001, two-sided t-test). The luciferase signals of cells with GAL4-DBD-TNRC18 fusion were first normalized to those of internal control (Renilla luciferase) and then normalized to those of cells with GAL4-DBD alone. The exact P value is shown in Supplementary Table 8..
    Figure Legend Snippet: a. Sanger sequencing to verify the CRISPR-Cas9-induced frameshift and KO of SETDB1 in HEK293 cells. b. Left: Western blot of SETDB1, TNRC18 and H3K9me3 in WT and SETDB1-KO HEK293 cells. Right: Western blot of SETDB1 in HEK293 cells with WT TNRC18, TNRC18 KO and TNRC18 W2858A mutation. Vinculin and Tubulin are the sample processing control. The representative results from 3 independent experiments are shown. c. Heatmap of TNRC18 CUT&RUN signal across ±5 kb at the TNRC18 peaks region in WT and SETDB1-KO HEK293 cells. d. Immunoblotting for H3K9me3, histone H3 acetylation (H3ac) and total H3 in HEK293 cells, either WT or with the homozygous mutation of TNRC18W2858A. Data shown are the representative results from 3 independent experiments. e. The heatmap of CUT&RUN signals of H3ac (left) and H3K9me3 (right) in HEK293 cells, either WT or with the homozygous mutation of TNRC18W2858A, across ±5 kb from the called peaks. f. Box plot showing the log2 values of normalized CUT&RUN sequencing counts in HEK293 cells with homozygous TNRC18W2858A mutation, compared to WT, at the indicated different TE classes. The boundaries of box plots indicate the 25th and 75th percentiles, the center line indicates the median, and the whiskers indicate 1.5× the interquartile range. Sample size of each box plot is listed in Supplementary Table 8. g. Global methylation levels of WT and TNRC18W2858A-mutated HEK293 cells. h. DNA methylation levels of WT and TNRC18W2858A-mutated HEK293 cells at SINE, LTR7Y, LTR12C and CpG_ island. The boundaries of box plots indicate the 25th and 75th percentiles, the center line indicates the median, and the whiskers indicate 1.5× the interquartile range. Sample size of each box plot is listed in Supplementary Table 8. i. The dual-luciferase reporter-based system for assaying the activity of full-length TNRC18 fused to the GAL4’s DNA binding domain (GAL4-DBD), relative to GAL4-DBD alone (n = 6 biologically independent samples; data presented as the mean ±s.d.; ****P < 0.0001, two-sided t-test). The luciferase signals of cells with GAL4-DBD-TNRC18 fusion were first normalized to those of internal control (Renilla luciferase) and then normalized to those of cells with GAL4-DBD alone. The exact P value is shown in Supplementary Table 8..

    Techniques Used: Sequencing, CRISPR, Western Blot, Mutagenesis, Methylation, DNA Methylation Assay, Luciferase, Activity Assay, Binding Assay

    ultra ii dna library prep kit  (New England Biolabs)


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    New England Biolabs ultra ii dna library prep kit
    Ultra Ii Dna Library Prep Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ultra ii dna library prep kit  (New England Biolabs)


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    New England Biolabs ultra ii dna library prep kit
    a, CasID identified ERV-bound proteins in HEK293 cells, with hits ranked by the fold change of normalized spectral abundance factor (NSAF) relative to control. Label font colors: green, zinc finger proteins; blue, histone and <t>DNA-methylation-related</t> factors; yellow, histone chaperones; purple, N6-methyladenosine-related factors; red, TNRC18. b,c, Scatter plots showing TE families exhibiting expression changes based on RNA-seq of HEK293 cells with endogenous TNRC18 KD (using TNRC18 shRNA (shTNRC18); b) relative to TNRC18 KD followed by TNRC18 re-expression (shTNRC18-rescue; n = 2 independent experiments), or cells with endogenous TNRC18 KO (TNRC18-KO; c) relative to WT (n = 3 independent experiments). The <t>significance</t> <t>cut-off</t> is the fold change in expression over 1.50 and adjusted P value less than 0.01 for transcripts with base mean read counts over 10. Adjusted P value was calculated using negative binomial model-based methods (DESeq2). d, RT–qPCR for TEs (top) and immunity-related genes (bottom) in HEK293 cells with shTNRC18 versus control shRNA (shControl) or shTNRC18- rescue, or TNRC18 KO versus WT (n = 3 independent experiments; plotted as mean ± s.d. after normalization to GAPDH and control samples). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-sided t-test. Exact P values are shown in Supplementary Table 8. e,f, GSEA revealed pathway enrichment in cells with shTNRC18 versus shControl (e), or TNRC18 KO versus WT (f). Immunity-related gene sets are labelled in red. The y axis and x axis show normalized enrichment score (NES) and false discovery rate (FDR) q values, respectively. g, RT–qPCR for TEs in the indicated TNRC18 KO cells versus WT cells (n = 3 independent experiments; plotted as the mean ± s.d. after normalization to GAPDH and to WT). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-sided t-test. Exact P values are shown in Supplementary Table 8. h,i, RNA-seq revealed TEs exhibiting expression changes in SNU-1 cells (h) and HT-29 cells (i) with TNRC18 KO versus WT (n = 3 independent experiments). Significance cut-off is the same as in b,c. Adjusted P values were calculated using negative binomial model-based methods (DESeq2).
    Ultra Ii Dna Library Prep Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ultra ii dna library prep kit/product/New England Biolabs
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ultra ii dna library prep kit - by Bioz Stars, 2024-07
    97/100 stars

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    1) Product Images from "TNRC18 engages H3K9me3 to mediate silencing of endogenous retrotransposons"

    Article Title: TNRC18 engages H3K9me3 to mediate silencing of endogenous retrotransposons

    Journal: Nature

    doi: 10.1038/s41586-023-06688-z

    a, CasID identified ERV-bound proteins in HEK293 cells, with hits ranked by the fold change of normalized spectral abundance factor (NSAF) relative to control. Label font colors: green, zinc finger proteins; blue, histone and DNA-methylation-related factors; yellow, histone chaperones; purple, N6-methyladenosine-related factors; red, TNRC18. b,c, Scatter plots showing TE families exhibiting expression changes based on RNA-seq of HEK293 cells with endogenous TNRC18 KD (using TNRC18 shRNA (shTNRC18); b) relative to TNRC18 KD followed by TNRC18 re-expression (shTNRC18-rescue; n = 2 independent experiments), or cells with endogenous TNRC18 KO (TNRC18-KO; c) relative to WT (n = 3 independent experiments). The significance cut-off is the fold change in expression over 1.50 and adjusted P value less than 0.01 for transcripts with base mean read counts over 10. Adjusted P value was calculated using negative binomial model-based methods (DESeq2). d, RT–qPCR for TEs (top) and immunity-related genes (bottom) in HEK293 cells with shTNRC18 versus control shRNA (shControl) or shTNRC18- rescue, or TNRC18 KO versus WT (n = 3 independent experiments; plotted as mean ± s.d. after normalization to GAPDH and control samples). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-sided t-test. Exact P values are shown in Supplementary Table 8. e,f, GSEA revealed pathway enrichment in cells with shTNRC18 versus shControl (e), or TNRC18 KO versus WT (f). Immunity-related gene sets are labelled in red. The y axis and x axis show normalized enrichment score (NES) and false discovery rate (FDR) q values, respectively. g, RT–qPCR for TEs in the indicated TNRC18 KO cells versus WT cells (n = 3 independent experiments; plotted as the mean ± s.d. after normalization to GAPDH and to WT). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-sided t-test. Exact P values are shown in Supplementary Table 8. h,i, RNA-seq revealed TEs exhibiting expression changes in SNU-1 cells (h) and HT-29 cells (i) with TNRC18 KO versus WT (n = 3 independent experiments). Significance cut-off is the same as in b,c. Adjusted P values were calculated using negative binomial model-based methods (DESeq2).
    Figure Legend Snippet: a, CasID identified ERV-bound proteins in HEK293 cells, with hits ranked by the fold change of normalized spectral abundance factor (NSAF) relative to control. Label font colors: green, zinc finger proteins; blue, histone and DNA-methylation-related factors; yellow, histone chaperones; purple, N6-methyladenosine-related factors; red, TNRC18. b,c, Scatter plots showing TE families exhibiting expression changes based on RNA-seq of HEK293 cells with endogenous TNRC18 KD (using TNRC18 shRNA (shTNRC18); b) relative to TNRC18 KD followed by TNRC18 re-expression (shTNRC18-rescue; n = 2 independent experiments), or cells with endogenous TNRC18 KO (TNRC18-KO; c) relative to WT (n = 3 independent experiments). The significance cut-off is the fold change in expression over 1.50 and adjusted P value less than 0.01 for transcripts with base mean read counts over 10. Adjusted P value was calculated using negative binomial model-based methods (DESeq2). d, RT–qPCR for TEs (top) and immunity-related genes (bottom) in HEK293 cells with shTNRC18 versus control shRNA (shControl) or shTNRC18- rescue, or TNRC18 KO versus WT (n = 3 independent experiments; plotted as mean ± s.d. after normalization to GAPDH and control samples). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-sided t-test. Exact P values are shown in Supplementary Table 8. e,f, GSEA revealed pathway enrichment in cells with shTNRC18 versus shControl (e), or TNRC18 KO versus WT (f). Immunity-related gene sets are labelled in red. The y axis and x axis show normalized enrichment score (NES) and false discovery rate (FDR) q values, respectively. g, RT–qPCR for TEs in the indicated TNRC18 KO cells versus WT cells (n = 3 independent experiments; plotted as the mean ± s.d. after normalization to GAPDH and to WT). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-sided t-test. Exact P values are shown in Supplementary Table 8. h,i, RNA-seq revealed TEs exhibiting expression changes in SNU-1 cells (h) and HT-29 cells (i) with TNRC18 KO versus WT (n = 3 independent experiments). Significance cut-off is the same as in b,c. Adjusted P values were calculated using negative binomial model-based methods (DESeq2).

    Techniques Used: DNA Methylation Assay, Expressing, RNA Sequencing Assay, shRNA, Quantitative RT-PCR

    a. Sanger sequencing to verify the CRISPR-Cas9-induced frameshift and KO of SETDB1 in HEK293 cells. b. Left: Western blot of SETDB1, TNRC18 and H3K9me3 in WT and SETDB1-KO HEK293 cells. Right: Western blot of SETDB1 in HEK293 cells with WT TNRC18, TNRC18 KO and TNRC18 W2858A mutation. Vinculin and Tubulin are the sample processing control. The representative results from 3 independent experiments are shown. c. Heatmap of TNRC18 CUT&RUN signal across ±5 kb at the TNRC18 peaks region in WT and SETDB1-KO HEK293 cells. d. Immunoblotting for H3K9me3, histone H3 acetylation (H3ac) and total H3 in HEK293 cells, either WT or with the homozygous mutation of TNRC18W2858A. Data shown are the representative results from 3 independent experiments. e. The heatmap of CUT&RUN signals of H3ac (left) and H3K9me3 (right) in HEK293 cells, either WT or with the homozygous mutation of TNRC18W2858A, across ±5 kb from the called peaks. f. Box plot showing the log2 values of normalized CUT&RUN sequencing counts in HEK293 cells with homozygous TNRC18W2858A mutation, compared to WT, at the indicated different TE classes. The boundaries of box plots indicate the 25th and 75th percentiles, the center line indicates the median, and the whiskers indicate 1.5× the interquartile range. Sample size of each box plot is listed in Supplementary Table 8. g. Global methylation levels of WT and TNRC18W2858A-mutated HEK293 cells. h. DNA methylation levels of WT and TNRC18W2858A-mutated HEK293 cells at SINE, LTR7Y, LTR12C and CpG_ island. The boundaries of box plots indicate the 25th and 75th percentiles, the center line indicates the median, and the whiskers indicate 1.5× the interquartile range. Sample size of each box plot is listed in Supplementary Table 8. i. The dual-luciferase reporter-based system for assaying the activity of full-length TNRC18 fused to the GAL4’s DNA binding domain (GAL4-DBD), relative to GAL4-DBD alone (n = 6 biologically independent samples; data presented as the mean ±s.d.; ****P < 0.0001, two-sided t-test). The luciferase signals of cells with GAL4-DBD-TNRC18 fusion were first normalized to those of internal control (Renilla luciferase) and then normalized to those of cells with GAL4-DBD alone. The exact P value is shown in Supplementary Table 8..
    Figure Legend Snippet: a. Sanger sequencing to verify the CRISPR-Cas9-induced frameshift and KO of SETDB1 in HEK293 cells. b. Left: Western blot of SETDB1, TNRC18 and H3K9me3 in WT and SETDB1-KO HEK293 cells. Right: Western blot of SETDB1 in HEK293 cells with WT TNRC18, TNRC18 KO and TNRC18 W2858A mutation. Vinculin and Tubulin are the sample processing control. The representative results from 3 independent experiments are shown. c. Heatmap of TNRC18 CUT&RUN signal across ±5 kb at the TNRC18 peaks region in WT and SETDB1-KO HEK293 cells. d. Immunoblotting for H3K9me3, histone H3 acetylation (H3ac) and total H3 in HEK293 cells, either WT or with the homozygous mutation of TNRC18W2858A. Data shown are the representative results from 3 independent experiments. e. The heatmap of CUT&RUN signals of H3ac (left) and H3K9me3 (right) in HEK293 cells, either WT or with the homozygous mutation of TNRC18W2858A, across ±5 kb from the called peaks. f. Box plot showing the log2 values of normalized CUT&RUN sequencing counts in HEK293 cells with homozygous TNRC18W2858A mutation, compared to WT, at the indicated different TE classes. The boundaries of box plots indicate the 25th and 75th percentiles, the center line indicates the median, and the whiskers indicate 1.5× the interquartile range. Sample size of each box plot is listed in Supplementary Table 8. g. Global methylation levels of WT and TNRC18W2858A-mutated HEK293 cells. h. DNA methylation levels of WT and TNRC18W2858A-mutated HEK293 cells at SINE, LTR7Y, LTR12C and CpG_ island. The boundaries of box plots indicate the 25th and 75th percentiles, the center line indicates the median, and the whiskers indicate 1.5× the interquartile range. Sample size of each box plot is listed in Supplementary Table 8. i. The dual-luciferase reporter-based system for assaying the activity of full-length TNRC18 fused to the GAL4’s DNA binding domain (GAL4-DBD), relative to GAL4-DBD alone (n = 6 biologically independent samples; data presented as the mean ±s.d.; ****P < 0.0001, two-sided t-test). The luciferase signals of cells with GAL4-DBD-TNRC18 fusion were first normalized to those of internal control (Renilla luciferase) and then normalized to those of cells with GAL4-DBD alone. The exact P value is shown in Supplementary Table 8..

    Techniques Used: Sequencing, CRISPR, Western Blot, Mutagenesis, Methylation, DNA Methylation Assay, Luciferase, Activity Assay, Binding Assay

    ultra ii dna library prep kit  (New England Biolabs)


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    New England Biolabs ultra ii dna library prep kit
    a, CasID identified ERV-bound proteins in HEK293 cells, with hits ranked by the fold change of normalized spectral abundance factor (NSAF) relative to control. Label font colors: green, zinc finger proteins; blue, histone and <t>DNA-methylation-related</t> factors; yellow, histone chaperones; purple, N6-methyladenosine-related factors; red, TNRC18. b,c, Scatter plots showing TE families exhibiting expression changes based on RNA-seq of HEK293 cells with endogenous TNRC18 KD (using TNRC18 shRNA (shTNRC18); b) relative to TNRC18 KD followed by TNRC18 re-expression (shTNRC18-rescue; n = 2 independent experiments), or cells with endogenous TNRC18 KO (TNRC18-KO; c) relative to WT (n = 3 independent experiments). The <t>significance</t> <t>cut-off</t> is the fold change in expression over 1.50 and adjusted P value less than 0.01 for transcripts with base mean read counts over 10. Adjusted P value was calculated using negative binomial model-based methods (DESeq2). d, RT–qPCR for TEs (top) and immunity-related genes (bottom) in HEK293 cells with shTNRC18 versus control shRNA (shControl) or shTNRC18- rescue, or TNRC18 KO versus WT (n = 3 independent experiments; plotted as mean ± s.d. after normalization to GAPDH and control samples). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-sided t-test. Exact P values are shown in Supplementary Table 8. e,f, GSEA revealed pathway enrichment in cells with shTNRC18 versus shControl (e), or TNRC18 KO versus WT (f). Immunity-related gene sets are labelled in red. The y axis and x axis show normalized enrichment score (NES) and false discovery rate (FDR) q values, respectively. g, RT–qPCR for TEs in the indicated TNRC18 KO cells versus WT cells (n = 3 independent experiments; plotted as the mean ± s.d. after normalization to GAPDH and to WT). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-sided t-test. Exact P values are shown in Supplementary Table 8. h,i, RNA-seq revealed TEs exhibiting expression changes in SNU-1 cells (h) and HT-29 cells (i) with TNRC18 KO versus WT (n = 3 independent experiments). Significance cut-off is the same as in b,c. Adjusted P values were calculated using negative binomial model-based methods (DESeq2).
    Ultra Ii Dna Library Prep Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    a, CasID identified ERV-bound proteins in HEK293 cells, with hits ranked by the fold change of normalized spectral abundance factor (NSAF) relative to control. Label font colors: green, zinc finger proteins; blue, histone and <t>DNA-methylation-related</t> factors; yellow, histone chaperones; purple, N6-methyladenosine-related factors; red, TNRC18. b,c, Scatter plots showing TE families exhibiting expression changes based on RNA-seq of HEK293 cells with endogenous TNRC18 KD (using TNRC18 shRNA (shTNRC18); b) relative to TNRC18 KD followed by TNRC18 re-expression (shTNRC18-rescue; n = 2 independent experiments), or cells with endogenous TNRC18 KO (TNRC18-KO; c) relative to WT (n = 3 independent experiments). The <t>significance</t> <t>cut-off</t> is the fold change in expression over 1.50 and adjusted P value less than 0.01 for transcripts with base mean read counts over 10. Adjusted P value was calculated using negative binomial model-based methods (DESeq2). d, RT–qPCR for TEs (top) and immunity-related genes (bottom) in HEK293 cells with shTNRC18 versus control shRNA (shControl) or shTNRC18- rescue, or TNRC18 KO versus WT (n = 3 independent experiments; plotted as mean ± s.d. after normalization to GAPDH and control samples). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-sided t-test. Exact P values are shown in Supplementary Table 8. e,f, GSEA revealed pathway enrichment in cells with shTNRC18 versus shControl (e), or TNRC18 KO versus WT (f). Immunity-related gene sets are labelled in red. The y axis and x axis show normalized enrichment score (NES) and false discovery rate (FDR) q values, respectively. g, RT–qPCR for TEs in the indicated TNRC18 KO cells versus WT cells (n = 3 independent experiments; plotted as the mean ± s.d. after normalization to GAPDH and to WT). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-sided t-test. Exact P values are shown in Supplementary Table 8. h,i, RNA-seq revealed TEs exhibiting expression changes in SNU-1 cells (h) and HT-29 cells (i) with TNRC18 KO versus WT (n = 3 independent experiments). Significance cut-off is the same as in b,c. Adjusted P values were calculated using negative binomial model-based methods (DESeq2).
    Nebnext Ultra Ii Dna Library Prep Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs nebnext ultra ii dna library prepwith sample purification beads
    a, CasID identified ERV-bound proteins in HEK293 cells, with hits ranked by the fold change of normalized spectral abundance factor (NSAF) relative to control. Label font colors: green, zinc finger proteins; blue, histone and <t>DNA-methylation-related</t> factors; yellow, histone chaperones; purple, N6-methyladenosine-related factors; red, TNRC18. b,c, Scatter plots showing TE families exhibiting expression changes based on RNA-seq of HEK293 cells with endogenous TNRC18 KD (using TNRC18 shRNA (shTNRC18); b) relative to TNRC18 KD followed by TNRC18 re-expression (shTNRC18-rescue; n = 2 independent experiments), or cells with endogenous TNRC18 KO (TNRC18-KO; c) relative to WT (n = 3 independent experiments). The <t>significance</t> <t>cut-off</t> is the fold change in expression over 1.50 and adjusted P value less than 0.01 for transcripts with base mean read counts over 10. Adjusted P value was calculated using negative binomial model-based methods (DESeq2). d, RT–qPCR for TEs (top) and immunity-related genes (bottom) in HEK293 cells with shTNRC18 versus control shRNA (shControl) or shTNRC18- rescue, or TNRC18 KO versus WT (n = 3 independent experiments; plotted as mean ± s.d. after normalization to GAPDH and control samples). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-sided t-test. Exact P values are shown in Supplementary Table 8. e,f, GSEA revealed pathway enrichment in cells with shTNRC18 versus shControl (e), or TNRC18 KO versus WT (f). Immunity-related gene sets are labelled in red. The y axis and x axis show normalized enrichment score (NES) and false discovery rate (FDR) q values, respectively. g, RT–qPCR for TEs in the indicated TNRC18 KO cells versus WT cells (n = 3 independent experiments; plotted as the mean ± s.d. after normalization to GAPDH and to WT). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-sided t-test. Exact P values are shown in Supplementary Table 8. h,i, RNA-seq revealed TEs exhibiting expression changes in SNU-1 cells (h) and HT-29 cells (i) with TNRC18 KO versus WT (n = 3 independent experiments). Significance cut-off is the same as in b,c. Adjusted P values were calculated using negative binomial model-based methods (DESeq2).
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    KEY RESOURCES TABLE

    Journal: Developmental cell

    Article Title: Transcription factor NFYa controls cardiomyocyte metabolism and proliferation during mouse fetal heart development

    doi: 10.1016/j.devcel.2023.10.012

    Figure Lengend Snippet: KEY RESOURCES TABLE

    Article Snippet: NEBNext Ultra II DNA Library Preparation Kit , NEB , Cat#E7103.

    Techniques: Produced, Western Blot, Virus, Recombinant, Modification, Protease Inhibitor, Expressing, Software

    a, CasID identified ERV-bound proteins in HEK293 cells, with hits ranked by the fold change of normalized spectral abundance factor (NSAF) relative to control. Label font colors: green, zinc finger proteins; blue, histone and DNA-methylation-related factors; yellow, histone chaperones; purple, N6-methyladenosine-related factors; red, TNRC18. b,c, Scatter plots showing TE families exhibiting expression changes based on RNA-seq of HEK293 cells with endogenous TNRC18 KD (using TNRC18 shRNA (shTNRC18); b) relative to TNRC18 KD followed by TNRC18 re-expression (shTNRC18-rescue; n = 2 independent experiments), or cells with endogenous TNRC18 KO (TNRC18-KO; c) relative to WT (n = 3 independent experiments). The significance cut-off is the fold change in expression over 1.50 and adjusted P value less than 0.01 for transcripts with base mean read counts over 10. Adjusted P value was calculated using negative binomial model-based methods (DESeq2). d, RT–qPCR for TEs (top) and immunity-related genes (bottom) in HEK293 cells with shTNRC18 versus control shRNA (shControl) or shTNRC18- rescue, or TNRC18 KO versus WT (n = 3 independent experiments; plotted as mean ± s.d. after normalization to GAPDH and control samples). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-sided t-test. Exact P values are shown in Supplementary Table 8. e,f, GSEA revealed pathway enrichment in cells with shTNRC18 versus shControl (e), or TNRC18 KO versus WT (f). Immunity-related gene sets are labelled in red. The y axis and x axis show normalized enrichment score (NES) and false discovery rate (FDR) q values, respectively. g, RT–qPCR for TEs in the indicated TNRC18 KO cells versus WT cells (n = 3 independent experiments; plotted as the mean ± s.d. after normalization to GAPDH and to WT). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-sided t-test. Exact P values are shown in Supplementary Table 8. h,i, RNA-seq revealed TEs exhibiting expression changes in SNU-1 cells (h) and HT-29 cells (i) with TNRC18 KO versus WT (n = 3 independent experiments). Significance cut-off is the same as in b,c. Adjusted P values were calculated using negative binomial model-based methods (DESeq2).

    Journal: Nature

    Article Title: TNRC18 engages H3K9me3 to mediate silencing of endogenous retrotransposons

    doi: 10.1038/s41586-023-06688-z

    Figure Lengend Snippet: a, CasID identified ERV-bound proteins in HEK293 cells, with hits ranked by the fold change of normalized spectral abundance factor (NSAF) relative to control. Label font colors: green, zinc finger proteins; blue, histone and DNA-methylation-related factors; yellow, histone chaperones; purple, N6-methyladenosine-related factors; red, TNRC18. b,c, Scatter plots showing TE families exhibiting expression changes based on RNA-seq of HEK293 cells with endogenous TNRC18 KD (using TNRC18 shRNA (shTNRC18); b) relative to TNRC18 KD followed by TNRC18 re-expression (shTNRC18-rescue; n = 2 independent experiments), or cells with endogenous TNRC18 KO (TNRC18-KO; c) relative to WT (n = 3 independent experiments). The significance cut-off is the fold change in expression over 1.50 and adjusted P value less than 0.01 for transcripts with base mean read counts over 10. Adjusted P value was calculated using negative binomial model-based methods (DESeq2). d, RT–qPCR for TEs (top) and immunity-related genes (bottom) in HEK293 cells with shTNRC18 versus control shRNA (shControl) or shTNRC18- rescue, or TNRC18 KO versus WT (n = 3 independent experiments; plotted as mean ± s.d. after normalization to GAPDH and control samples). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-sided t-test. Exact P values are shown in Supplementary Table 8. e,f, GSEA revealed pathway enrichment in cells with shTNRC18 versus shControl (e), or TNRC18 KO versus WT (f). Immunity-related gene sets are labelled in red. The y axis and x axis show normalized enrichment score (NES) and false discovery rate (FDR) q values, respectively. g, RT–qPCR for TEs in the indicated TNRC18 KO cells versus WT cells (n = 3 independent experiments; plotted as the mean ± s.d. after normalization to GAPDH and to WT). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-sided t-test. Exact P values are shown in Supplementary Table 8. h,i, RNA-seq revealed TEs exhibiting expression changes in SNU-1 cells (h) and HT-29 cells (i) with TNRC18 KO versus WT (n = 3 independent experiments). Significance cut-off is the same as in b,c. Adjusted P values were calculated using negative binomial model-based methods (DESeq2).

    Article Snippet: Next, 10 ng of the purified CUT&RUN DNA was used for preparation of multiplexed libraries with a NEB Ultra II DNA Library Prep kit per the manufacturer’s instruction (NEB, E7103).

    Techniques: DNA Methylation Assay, Expressing, RNA Sequencing Assay, shRNA, Quantitative RT-PCR

    a. Sanger sequencing to verify the CRISPR-Cas9-induced frameshift and KO of SETDB1 in HEK293 cells. b. Left: Western blot of SETDB1, TNRC18 and H3K9me3 in WT and SETDB1-KO HEK293 cells. Right: Western blot of SETDB1 in HEK293 cells with WT TNRC18, TNRC18 KO and TNRC18 W2858A mutation. Vinculin and Tubulin are the sample processing control. The representative results from 3 independent experiments are shown. c. Heatmap of TNRC18 CUT&RUN signal across ±5 kb at the TNRC18 peaks region in WT and SETDB1-KO HEK293 cells. d. Immunoblotting for H3K9me3, histone H3 acetylation (H3ac) and total H3 in HEK293 cells, either WT or with the homozygous mutation of TNRC18W2858A. Data shown are the representative results from 3 independent experiments. e. The heatmap of CUT&RUN signals of H3ac (left) and H3K9me3 (right) in HEK293 cells, either WT or with the homozygous mutation of TNRC18W2858A, across ±5 kb from the called peaks. f. Box plot showing the log2 values of normalized CUT&RUN sequencing counts in HEK293 cells with homozygous TNRC18W2858A mutation, compared to WT, at the indicated different TE classes. The boundaries of box plots indicate the 25th and 75th percentiles, the center line indicates the median, and the whiskers indicate 1.5× the interquartile range. Sample size of each box plot is listed in Supplementary Table 8. g. Global methylation levels of WT and TNRC18W2858A-mutated HEK293 cells. h. DNA methylation levels of WT and TNRC18W2858A-mutated HEK293 cells at SINE, LTR7Y, LTR12C and CpG_ island. The boundaries of box plots indicate the 25th and 75th percentiles, the center line indicates the median, and the whiskers indicate 1.5× the interquartile range. Sample size of each box plot is listed in Supplementary Table 8. i. The dual-luciferase reporter-based system for assaying the activity of full-length TNRC18 fused to the GAL4’s DNA binding domain (GAL4-DBD), relative to GAL4-DBD alone (n = 6 biologically independent samples; data presented as the mean ±s.d.; ****P < 0.0001, two-sided t-test). The luciferase signals of cells with GAL4-DBD-TNRC18 fusion were first normalized to those of internal control (Renilla luciferase) and then normalized to those of cells with GAL4-DBD alone. The exact P value is shown in Supplementary Table 8..

    Journal: Nature

    Article Title: TNRC18 engages H3K9me3 to mediate silencing of endogenous retrotransposons

    doi: 10.1038/s41586-023-06688-z

    Figure Lengend Snippet: a. Sanger sequencing to verify the CRISPR-Cas9-induced frameshift and KO of SETDB1 in HEK293 cells. b. Left: Western blot of SETDB1, TNRC18 and H3K9me3 in WT and SETDB1-KO HEK293 cells. Right: Western blot of SETDB1 in HEK293 cells with WT TNRC18, TNRC18 KO and TNRC18 W2858A mutation. Vinculin and Tubulin are the sample processing control. The representative results from 3 independent experiments are shown. c. Heatmap of TNRC18 CUT&RUN signal across ±5 kb at the TNRC18 peaks region in WT and SETDB1-KO HEK293 cells. d. Immunoblotting for H3K9me3, histone H3 acetylation (H3ac) and total H3 in HEK293 cells, either WT or with the homozygous mutation of TNRC18W2858A. Data shown are the representative results from 3 independent experiments. e. The heatmap of CUT&RUN signals of H3ac (left) and H3K9me3 (right) in HEK293 cells, either WT or with the homozygous mutation of TNRC18W2858A, across ±5 kb from the called peaks. f. Box plot showing the log2 values of normalized CUT&RUN sequencing counts in HEK293 cells with homozygous TNRC18W2858A mutation, compared to WT, at the indicated different TE classes. The boundaries of box plots indicate the 25th and 75th percentiles, the center line indicates the median, and the whiskers indicate 1.5× the interquartile range. Sample size of each box plot is listed in Supplementary Table 8. g. Global methylation levels of WT and TNRC18W2858A-mutated HEK293 cells. h. DNA methylation levels of WT and TNRC18W2858A-mutated HEK293 cells at SINE, LTR7Y, LTR12C and CpG_ island. The boundaries of box plots indicate the 25th and 75th percentiles, the center line indicates the median, and the whiskers indicate 1.5× the interquartile range. Sample size of each box plot is listed in Supplementary Table 8. i. The dual-luciferase reporter-based system for assaying the activity of full-length TNRC18 fused to the GAL4’s DNA binding domain (GAL4-DBD), relative to GAL4-DBD alone (n = 6 biologically independent samples; data presented as the mean ±s.d.; ****P < 0.0001, two-sided t-test). The luciferase signals of cells with GAL4-DBD-TNRC18 fusion were first normalized to those of internal control (Renilla luciferase) and then normalized to those of cells with GAL4-DBD alone. The exact P value is shown in Supplementary Table 8..

    Article Snippet: Next, 10 ng of the purified CUT&RUN DNA was used for preparation of multiplexed libraries with a NEB Ultra II DNA Library Prep kit per the manufacturer’s instruction (NEB, E7103).

    Techniques: Sequencing, CRISPR, Western Blot, Mutagenesis, Methylation, DNA Methylation Assay, Luciferase, Activity Assay, Binding Assay