ultra rna library prep kit  (New England Biolabs)


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    Name:
    NEBNext Ultra RNA Library Prep Kit for Illumina
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    E7530L
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    Structured Review

    New England Biolabs ultra rna library prep kit
    SIRT7 is involved in pre-rRNA processing. ( a ) Knockdown of SIRT7 impairs pre-rRNA synthesis and processing in vivo . U2OS cells transfected with control (siCtrl) or SIRT7-specific siRNAs (siSIRT7) were metabolically labelled with 3 H-uridine. <t>RNA</t> was analysed by agarose gel electrophoresis and fluorography. The bar diagram shows quantification of the processing intermediates, values from siCtrl cells being set to 1. ( b ) In vitro processing assay. Extracts from L1210 cells were incubated with 32 P-labelled RNA comprising the 5′ETS depicted in the scheme above. 32 P-labelled RNA and cleavage products were analysed by gel electrophoresis and PhosphorImaging. See also Supplementary Fig. 3a . ( c ) 5′ETS processing is inhibited by NAM. The assay contained radiolabelled RNA (+541/+1290) and extracts from L1210 cells cultured for 6 h in the absence or presence of NAM. ( d ) Processing is enhanced by NAD + . Processing assays containing radiolabelled RNA (+541/+1290) were substituted with NAD + as indicated. ( e ) The catalytic activity of SIRT7 is required for pre-rRNA cleavage. Assays were supplemented with 15 or 30 ng of purified wildtype (WT) or mutant (H187Y) Flag-SIRT7 ( Supplementary Fig. 3b ). ( f ) Depletion of SIRT7 impairs processing. SIRT7 was depleted from L1210 cells by shRNAs (shSIRT7-1, shSIRT7-2, Supplementary Fig. 3c ). Extracts from non-infected cells (−) or cells expressing control shRNA (shCtrl) served as control (left). To rescue impaired cleavage, 15 ng of wild-type Flag-SIRT7 (WT) or mutant H187Y (HY) were added to SIRT7-depleted extracts (right). ( g ) Depletion of U3 snoRNA abolishes processing. U3 snoRNA was depleted by preincubating extracts with U3-specific antisense <t>oligos</t> (ASO, 50 ng μl −1 ) and 2 U of RNase H ( Supplementary Fig. 3d ). In vitro processing was performed with undepleted (−) or depleted extracts in the absence or presence of 15 ng Flag-SIRT7. Bar diagrams in c – g show quantification of the ratio of cleaved versus uncleaved transcripts, presented as mean±s.d. from three independent experiments (* P

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    Images

    1) Product Images from "SIRT7-dependent deacetylation of the U3-55k protein controls pre-rRNA processing"

    Article Title: SIRT7-dependent deacetylation of the U3-55k protein controls pre-rRNA processing

    Journal: Nature Communications

    doi: 10.1038/ncomms10734

    SIRT7 is involved in pre-rRNA processing. ( a ) Knockdown of SIRT7 impairs pre-rRNA synthesis and processing in vivo . U2OS cells transfected with control (siCtrl) or SIRT7-specific siRNAs (siSIRT7) were metabolically labelled with 3 H-uridine. RNA was analysed by agarose gel electrophoresis and fluorography. The bar diagram shows quantification of the processing intermediates, values from siCtrl cells being set to 1. ( b ) In vitro processing assay. Extracts from L1210 cells were incubated with 32 P-labelled RNA comprising the 5′ETS depicted in the scheme above. 32 P-labelled RNA and cleavage products were analysed by gel electrophoresis and PhosphorImaging. See also Supplementary Fig. 3a . ( c ) 5′ETS processing is inhibited by NAM. The assay contained radiolabelled RNA (+541/+1290) and extracts from L1210 cells cultured for 6 h in the absence or presence of NAM. ( d ) Processing is enhanced by NAD + . Processing assays containing radiolabelled RNA (+541/+1290) were substituted with NAD + as indicated. ( e ) The catalytic activity of SIRT7 is required for pre-rRNA cleavage. Assays were supplemented with 15 or 30 ng of purified wildtype (WT) or mutant (H187Y) Flag-SIRT7 ( Supplementary Fig. 3b ). ( f ) Depletion of SIRT7 impairs processing. SIRT7 was depleted from L1210 cells by shRNAs (shSIRT7-1, shSIRT7-2, Supplementary Fig. 3c ). Extracts from non-infected cells (−) or cells expressing control shRNA (shCtrl) served as control (left). To rescue impaired cleavage, 15 ng of wild-type Flag-SIRT7 (WT) or mutant H187Y (HY) were added to SIRT7-depleted extracts (right). ( g ) Depletion of U3 snoRNA abolishes processing. U3 snoRNA was depleted by preincubating extracts with U3-specific antisense oligos (ASO, 50 ng μl −1 ) and 2 U of RNase H ( Supplementary Fig. 3d ). In vitro processing was performed with undepleted (−) or depleted extracts in the absence or presence of 15 ng Flag-SIRT7. Bar diagrams in c – g show quantification of the ratio of cleaved versus uncleaved transcripts, presented as mean±s.d. from three independent experiments (* P
    Figure Legend Snippet: SIRT7 is involved in pre-rRNA processing. ( a ) Knockdown of SIRT7 impairs pre-rRNA synthesis and processing in vivo . U2OS cells transfected with control (siCtrl) or SIRT7-specific siRNAs (siSIRT7) were metabolically labelled with 3 H-uridine. RNA was analysed by agarose gel electrophoresis and fluorography. The bar diagram shows quantification of the processing intermediates, values from siCtrl cells being set to 1. ( b ) In vitro processing assay. Extracts from L1210 cells were incubated with 32 P-labelled RNA comprising the 5′ETS depicted in the scheme above. 32 P-labelled RNA and cleavage products were analysed by gel electrophoresis and PhosphorImaging. See also Supplementary Fig. 3a . ( c ) 5′ETS processing is inhibited by NAM. The assay contained radiolabelled RNA (+541/+1290) and extracts from L1210 cells cultured for 6 h in the absence or presence of NAM. ( d ) Processing is enhanced by NAD + . Processing assays containing radiolabelled RNA (+541/+1290) were substituted with NAD + as indicated. ( e ) The catalytic activity of SIRT7 is required for pre-rRNA cleavage. Assays were supplemented with 15 or 30 ng of purified wildtype (WT) or mutant (H187Y) Flag-SIRT7 ( Supplementary Fig. 3b ). ( f ) Depletion of SIRT7 impairs processing. SIRT7 was depleted from L1210 cells by shRNAs (shSIRT7-1, shSIRT7-2, Supplementary Fig. 3c ). Extracts from non-infected cells (−) or cells expressing control shRNA (shCtrl) served as control (left). To rescue impaired cleavage, 15 ng of wild-type Flag-SIRT7 (WT) or mutant H187Y (HY) were added to SIRT7-depleted extracts (right). ( g ) Depletion of U3 snoRNA abolishes processing. U3 snoRNA was depleted by preincubating extracts with U3-specific antisense oligos (ASO, 50 ng μl −1 ) and 2 U of RNase H ( Supplementary Fig. 3d ). In vitro processing was performed with undepleted (−) or depleted extracts in the absence or presence of 15 ng Flag-SIRT7. Bar diagrams in c – g show quantification of the ratio of cleaved versus uncleaved transcripts, presented as mean±s.d. from three independent experiments (* P

    Techniques Used: In Vivo, Transfection, Metabolic Labelling, Agarose Gel Electrophoresis, In Vitro, Incubation, Nucleic Acid Electrophoresis, Cell Culture, Activity Assay, Purification, Mutagenesis, Infection, Expressing, shRNA, Allele-specific Oligonucleotide

    SIRT7 is associated with pre-rRNA and snoRNAs. ( a ) SIRT7 CLIP-seq reads mapped to a custom annotation file of a human rDNA repeat (middle) or the transcribed region (bottom). The region encoding 18S, 5.8S and 28S rRNA is highlighted. SIRT7 reads after subtraction of IgG reads were normalized to input reads ( y axis). ( b ) Gene ontology categories of SIRT7 CLIP-seq peaks. The most representative clusters are shown according to the ajusted P value (−log 10 ). ( c ) SIRT7-bound snoRNAs comprise C/D box, H/ACA box snoRNAs and scaRNAs. The number ( n ) and relative abundance (%) of each snoRNA class associated with SIRT7 is presented. ( d ) U3, SNORA73A and 73B snoRNAs are overrepresented among SIRT7-associated snoRNAs. SIRT7 reads mapped to corresponding snoRNAs are indicated as percentage of all snoRNAs identified by CLIP-seq. ( e ) Comparison of SIRT7-associated RNAs under native and denaturing conditions. His/V5-tagged SIRT7 expressed in HEK293T cells was affinity-purified on Ni-NTA-agarose under native or denaturing conditions, and associated RNAs were detected by RT–qPCR. Lysates from non-transfected HEK293T cells were used for control (Ctrl). Associated pre-RNA was monitored by RT–qPCR using primer H1 ( Supplementary Table 3 ). Bars represent means±s.d. from three experiments. See also Supplementary Fig. 2b,d . ( f ) ChIP assays showing association of endogenous SIRT7 (left panel) or transiently overexpressed Flag-SIRT7 (right panel) with the indicated gene loci in HEK293T cells. rDNA was amplified using primers H4 (coding) and H18 (IGS; Supplementary Table 3 ). Bars represent means±s.d. from three experiments. See also Supplementary Fig. 2d .
    Figure Legend Snippet: SIRT7 is associated with pre-rRNA and snoRNAs. ( a ) SIRT7 CLIP-seq reads mapped to a custom annotation file of a human rDNA repeat (middle) or the transcribed region (bottom). The region encoding 18S, 5.8S and 28S rRNA is highlighted. SIRT7 reads after subtraction of IgG reads were normalized to input reads ( y axis). ( b ) Gene ontology categories of SIRT7 CLIP-seq peaks. The most representative clusters are shown according to the ajusted P value (−log 10 ). ( c ) SIRT7-bound snoRNAs comprise C/D box, H/ACA box snoRNAs and scaRNAs. The number ( n ) and relative abundance (%) of each snoRNA class associated with SIRT7 is presented. ( d ) U3, SNORA73A and 73B snoRNAs are overrepresented among SIRT7-associated snoRNAs. SIRT7 reads mapped to corresponding snoRNAs are indicated as percentage of all snoRNAs identified by CLIP-seq. ( e ) Comparison of SIRT7-associated RNAs under native and denaturing conditions. His/V5-tagged SIRT7 expressed in HEK293T cells was affinity-purified on Ni-NTA-agarose under native or denaturing conditions, and associated RNAs were detected by RT–qPCR. Lysates from non-transfected HEK293T cells were used for control (Ctrl). Associated pre-RNA was monitored by RT–qPCR using primer H1 ( Supplementary Table 3 ). Bars represent means±s.d. from three experiments. See also Supplementary Fig. 2b,d . ( f ) ChIP assays showing association of endogenous SIRT7 (left panel) or transiently overexpressed Flag-SIRT7 (right panel) with the indicated gene loci in HEK293T cells. rDNA was amplified using primers H4 (coding) and H18 (IGS; Supplementary Table 3 ). Bars represent means±s.d. from three experiments. See also Supplementary Fig. 2d .

    Techniques Used: Cross-linking Immunoprecipitation, Affinity Purification, Quantitative RT-PCR, Transfection, Chromatin Immunoprecipitation, Amplification

    Pre-rRNA transcription and processing are attenuated under stress. ( a ) Northern blot of pre-rRNA and processing intermediates from HEK293T cells that were untreated, exposed to hyperosmotic stress for 90 min (hypertonic), or recovered to regular medium for 60 min (hypertonic rel.). Membranes were probed with 32 P-labelled antisense riboprobe specific to 47S pre-rRNA (5'ETS, top) or with ITS1 oligos hybridizing to pre-rRNA intermediates (middle panel). ( b ) Acetylation of U3-55k is increased on different cellular stress conditions. HEK293T cells expressing Flag-U3-55k were treated with actinomycin D (Act D, 0.1 μg ml −1 , 4 h), AICAR (0.5 mM, 12 h) or exposed to hypertonic stress. Acetylation of immunopurified Flag-U3-55k and equal loading was monitored on western blots using anti-pan-AcK and anti-Flag antibodies. ( c ) Cellular localization of SIRT7 and U3-55k on hyperosmotic stress. Images showing localization of GFP-U3-55k and SIRT7 in normal conditions and on exposure to hyperosmotic stress for 90 min. Nuclei were stained with Hoechst 33342. Scale bars, 10 μm. ( d ) Overexpression of SIRT7 alleviates processing defects on hypertonic stress. Northern blot of RNA from parental U2OS cells and from cells which stably express GFP-SIRT7 (U2OS-GFP-SIRT7) using 5′ETS and ITS1 probes as in a . ( e ) CLIP-RT–qPCR monitoring binding of Flag-U3-55k to pre-rRNA, U3 snoRNA and U2 snRNA in HEK293T cells cultured in normo-osmotic medium or exposed to hypertonic stress for 90 min. Precipitated RNA was analysed by RT–qPCR using the indicated primers. Bars represent the means±s.d. from three biological repeats (* P
    Figure Legend Snippet: Pre-rRNA transcription and processing are attenuated under stress. ( a ) Northern blot of pre-rRNA and processing intermediates from HEK293T cells that were untreated, exposed to hyperosmotic stress for 90 min (hypertonic), or recovered to regular medium for 60 min (hypertonic rel.). Membranes were probed with 32 P-labelled antisense riboprobe specific to 47S pre-rRNA (5'ETS, top) or with ITS1 oligos hybridizing to pre-rRNA intermediates (middle panel). ( b ) Acetylation of U3-55k is increased on different cellular stress conditions. HEK293T cells expressing Flag-U3-55k were treated with actinomycin D (Act D, 0.1 μg ml −1 , 4 h), AICAR (0.5 mM, 12 h) or exposed to hypertonic stress. Acetylation of immunopurified Flag-U3-55k and equal loading was monitored on western blots using anti-pan-AcK and anti-Flag antibodies. ( c ) Cellular localization of SIRT7 and U3-55k on hyperosmotic stress. Images showing localization of GFP-U3-55k and SIRT7 in normal conditions and on exposure to hyperosmotic stress for 90 min. Nuclei were stained with Hoechst 33342. Scale bars, 10 μm. ( d ) Overexpression of SIRT7 alleviates processing defects on hypertonic stress. Northern blot of RNA from parental U2OS cells and from cells which stably express GFP-SIRT7 (U2OS-GFP-SIRT7) using 5′ETS and ITS1 probes as in a . ( e ) CLIP-RT–qPCR monitoring binding of Flag-U3-55k to pre-rRNA, U3 snoRNA and U2 snRNA in HEK293T cells cultured in normo-osmotic medium or exposed to hypertonic stress for 90 min. Precipitated RNA was analysed by RT–qPCR using the indicated primers. Bars represent the means±s.d. from three biological repeats (* P

    Techniques Used: Northern Blot, Expressing, Activated Clotting Time Assay, Western Blot, Staining, Over Expression, Stable Transfection, Cross-linking Immunoprecipitation, Quantitative RT-PCR, Binding Assay, Cell Culture

    2) Product Images from "Evolution of a Designed Protein Assembly Encapsulating its Own RNA Genome"

    Article Title: Evolution of a Designed Protein Assembly Encapsulating its Own RNA Genome

    Journal:

    doi: 10.1038/nature25157

    Evolution and performance of nucleocapsids with exterior surface mutations in vitro or in vivo a . Heatmap of log enrichments between the injected pool and RNA recovered from the tail vein 60 minutes later. Purple and orange indicate mutations that were depleted or enriched in the selected population, respectively. Blue squares and black dots indicate the I53-50-v3 starting sequence and I53-50-v4 selected sequence, respectively. Residues not in the designed combinatorial library are colored gray. Note the strong enrichment of the E67K mutation and corresponding depletion of the native E67 allele. b . Design model of I53-50-v4. Coloring is as described in . c . Four variants were tested: a consensus sequence based on the most common residue at each position after selection in murine circulation (Consensus, I53-50-v4), the full length sequence with the greatest fold increase in population fraction (Most_enriched), the sequence with the most total counts (Top_count), and I53-50-v3 with only the E67K mutation (v3_E67K). Previous versions (I53-50-v1 through I53-50-v3) were also included as benchmarks. Each variant was individually expressed and purified by IMAC before being pooled (equal protein concentration) and purified en masse by SEC. The resulting nucleocapsid pool was then incubated in whole blood (n = 3 independent reactions). RNA was recovered at the indicated time points, and the fraction of each variant was determined by Illumina MiSeq counts taken at each time point. d . The same nucleocapsid pool used in ( c ) was injected retro-orbitally into mice (n = 5 biologically independent mice). I53-50-v3 was evaluated with (v3) and without (v3H) the H6Q and H9Q mutations, and both variants were found to have similar behavior. Error bars represent standard error of the mean. Fig. 1a
    Figure Legend Snippet: Evolution and performance of nucleocapsids with exterior surface mutations in vitro or in vivo a . Heatmap of log enrichments between the injected pool and RNA recovered from the tail vein 60 minutes later. Purple and orange indicate mutations that were depleted or enriched in the selected population, respectively. Blue squares and black dots indicate the I53-50-v3 starting sequence and I53-50-v4 selected sequence, respectively. Residues not in the designed combinatorial library are colored gray. Note the strong enrichment of the E67K mutation and corresponding depletion of the native E67 allele. b . Design model of I53-50-v4. Coloring is as described in . c . Four variants were tested: a consensus sequence based on the most common residue at each position after selection in murine circulation (Consensus, I53-50-v4), the full length sequence with the greatest fold increase in population fraction (Most_enriched), the sequence with the most total counts (Top_count), and I53-50-v3 with only the E67K mutation (v3_E67K). Previous versions (I53-50-v1 through I53-50-v3) were also included as benchmarks. Each variant was individually expressed and purified by IMAC before being pooled (equal protein concentration) and purified en masse by SEC. The resulting nucleocapsid pool was then incubated in whole blood (n = 3 independent reactions). RNA was recovered at the indicated time points, and the fraction of each variant was determined by Illumina MiSeq counts taken at each time point. d . The same nucleocapsid pool used in ( c ) was injected retro-orbitally into mice (n = 5 biologically independent mice). I53-50-v3 was evaluated with (v3) and without (v3H) the H6Q and H9Q mutations, and both variants were found to have similar behavior. Error bars represent standard error of the mean. Fig. 1a

    Techniques Used: In Vitro, In Vivo, Injection, Sequencing, Mutagenesis, Selection, Variant Assay, Purification, Protein Concentration, Size-exclusion Chromatography, Incubation, Mouse Assay

    Evolution and performance of nucleocapsids modified with hydrophilic polypeptides in vitro or in vivo a . The change in population fraction corresponding to each variant was calculated from Illumina MiSeq counts for the input pool (t = 0), RNA recovered from circulation after 30 minutes (n = 3 biologically independent mice), and RNA recovered from circulation after 60 minutes (n = 2 biologically independent mice). b . Scatter plot of log 10 enrichment of each hydrophilic polypeptide versus its net charge as calculated from the total number of charged residues in its sequence. c . Scatter plot of log 10 enrichment of each polypeptide versus the number of unique amino acids in its sequence. d . Each of 11 variants were individually expressed and purified by IMAC before being pooled (equal protein concentration) and purified en masse by SEC. The resulting nucleocapsid pool was then incubated in heparinized whole blood at 37 ° C (n = 3 independent reactions per time point). RNA was recovered at the indicated time points, and the fraction of each variant was determined by Illumina MiSeq counts taken at each time point. e . The same nucleocapsid pool used in ( d ) was injected retro-orbitally into mice (n = 5 biologically independent mice). RNA content was then assessed as in ( d ) using RNA isolated from tail vein draws at the indicated time points. All variants exhibit high stability in blood; however, the unmodified I53-50-v3 nucleocapsid (no polypeptide, blue) and a negative control polypeptide (ESESG, red) are cleared rapidly from circulation in vivo . Error bars represent standard error of the mean.
    Figure Legend Snippet: Evolution and performance of nucleocapsids modified with hydrophilic polypeptides in vitro or in vivo a . The change in population fraction corresponding to each variant was calculated from Illumina MiSeq counts for the input pool (t = 0), RNA recovered from circulation after 30 minutes (n = 3 biologically independent mice), and RNA recovered from circulation after 60 minutes (n = 2 biologically independent mice). b . Scatter plot of log 10 enrichment of each hydrophilic polypeptide versus its net charge as calculated from the total number of charged residues in its sequence. c . Scatter plot of log 10 enrichment of each polypeptide versus the number of unique amino acids in its sequence. d . Each of 11 variants were individually expressed and purified by IMAC before being pooled (equal protein concentration) and purified en masse by SEC. The resulting nucleocapsid pool was then incubated in heparinized whole blood at 37 ° C (n = 3 independent reactions per time point). RNA was recovered at the indicated time points, and the fraction of each variant was determined by Illumina MiSeq counts taken at each time point. e . The same nucleocapsid pool used in ( d ) was injected retro-orbitally into mice (n = 5 biologically independent mice). RNA content was then assessed as in ( d ) using RNA isolated from tail vein draws at the indicated time points. All variants exhibit high stability in blood; however, the unmodified I53-50-v3 nucleocapsid (no polypeptide, blue) and a negative control polypeptide (ESESG, red) are cleared rapidly from circulation in vivo . Error bars represent standard error of the mean.

    Techniques Used: Modification, In Vitro, In Vivo, Variant Assay, Mouse Assay, Sequencing, Purification, Protein Concentration, Size-exclusion Chromatography, Incubation, Injection, Isolation, Negative Control

    3) Product Images from "The m6A Reader ECT2 Controls Trichome Morphology by Affecting mRNA Stability in Arabidopsis"

    Article Title: The m6A Reader ECT2 Controls Trichome Morphology by Affecting mRNA Stability in Arabidopsis

    Journal:

    doi: 10.1105/tpc.17.00934

    ECT2 Is an m6 A Reader Protein Whose m6 A Binding Function Is Required for Normal Trichome Morphology. (A) Cryo-SEM analysis of trichome morphology in wild-type, ect2-1 , ect2-2 , ECT2pro :ECT2-Flag/ect2-1 , and ECT2pro :ECT2m-Flag/ect2-1 plants. Trichomes are from the third and fourth leaves of 3-week-old Arabidopsis plants. Bar = 100 μm. (B) Statistical analysis of trichome branch number in wild-type, ect2-1 , ect2-2 , ECT2pro :ECT2-Flag/ect2-1 , and ECT2pro :ECT2m-Flag/ect2-1 plants. Three hundred trichomes from the third and fourth leaves of 3-week-old Arabidopsis plants were analyzed. (C) In vitro RIP-LC-MS/MS assay showing that m6 A modification is enriched in GST-ECT2-bound mRNA compared with the flow-through and input samples. GST-ECT2 was expressed in E. coli , and mRNA was isolated from 14-d-old wild-type Arabidopsis seedlings. Data are presented as means ± se , n = 3 biological replicates × 2 technical replicates. ****P < 0.0001 by t test (two-sided). (D) In vivo FA-RIP-LC-MS/MS showing that m6 A modification is enriched in ECT2-Flag-bound RNA compared with IgG-bound RNA. Fourteen-day-old ECT2pro :ECT2-Flag/ect2-1 seedlings were used for the experiment. Data are presented as means ± se , n = 2 biological replicates × 3 technical replicates. **P < 0.01 by t test (two-sided).
    Figure Legend Snippet: ECT2 Is an m6 A Reader Protein Whose m6 A Binding Function Is Required for Normal Trichome Morphology. (A) Cryo-SEM analysis of trichome morphology in wild-type, ect2-1 , ect2-2 , ECT2pro :ECT2-Flag/ect2-1 , and ECT2pro :ECT2m-Flag/ect2-1 plants. Trichomes are from the third and fourth leaves of 3-week-old Arabidopsis plants. Bar = 100 μm. (B) Statistical analysis of trichome branch number in wild-type, ect2-1 , ect2-2 , ECT2pro :ECT2-Flag/ect2-1 , and ECT2pro :ECT2m-Flag/ect2-1 plants. Three hundred trichomes from the third and fourth leaves of 3-week-old Arabidopsis plants were analyzed. (C) In vitro RIP-LC-MS/MS assay showing that m6 A modification is enriched in GST-ECT2-bound mRNA compared with the flow-through and input samples. GST-ECT2 was expressed in E. coli , and mRNA was isolated from 14-d-old wild-type Arabidopsis seedlings. Data are presented as means ± se , n = 3 biological replicates × 2 technical replicates. ****P < 0.0001 by t test (two-sided). (D) In vivo FA-RIP-LC-MS/MS showing that m6 A modification is enriched in ECT2-Flag-bound RNA compared with IgG-bound RNA. Fourteen-day-old ECT2pro :ECT2-Flag/ect2-1 seedlings were used for the experiment. Data are presented as means ± se , n = 2 biological replicates × 3 technical replicates. **P < 0.01 by t test (two-sided).

    Techniques Used: Binding Assay, In Vitro, Liquid Chromatography, Mass Spectrometry, Modification, Flow Cytometry, Isolation, In Vivo

    4) Product Images from "The transcription factor Foxo1 controls germinal center B cell proliferation in response to T cell help"

    Article Title: The transcription factor Foxo1 controls germinal center B cell proliferation in response to T cell help

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20161263

    Foxo1 is required for GC maintenance. (A) Schematic illustration of the experimental protocol for B–D, F, and G. (B) Flow cytometry of NP-specific donor B cells (CD45.1 + B220 + NP + ). (C) Histograms representing the number of donor IgG1 − GC B cells (CD45.1 + B220 + NP + CD38 − IgG1 − ) and IgG1 + GC B cells (CD45.1 + B220 + NP + CD38 − IgG1 + ) in 10 6 splenocytes (left), and the ratio of DZ:LZ cells (right). n = 3 biological replicates. (D, left) DNA content measurement of Foxo1 +/+ and Foxo1 f/f LZ GC B cells assessed by 7-AAD staining. n = 5 and 3 biological replicates for tamoxifen and vehicle treatment, respectively. (right) Proliferation status of Foxo1 +/+ and Foxo1 f/f LZ GC B cells assessed by EdU incorporation 30 min after an EdU injection. n = 3 biological replicates. (E) Immunohistochemical analysis. (top) Schematic illustration of the experimental protocol. (bottom left) Representative images of immunofluorescence microscopy of spleen sections showing expression of CD45.1 ( Foxo1 f/f -derived donor cells), CD35 (FDC network), and IgD (follicular B cells). DZ and LZ defined by the presence of CD35 + FDCs are surrounded by dashed lines. Bars, 100 µm. (bottom right) Quantification of relative CD45.1 signal intensity in the DZ compared with that in the LZ. Each symbol represents a single GC, and red bars indicate the mean. n = 43 (tamoxifen) and 40 (vehicle) GC pooled from three animals. (F) Hierarchical clustering of the gene expression profiles of Foxo1 +/+ DZ, Foxo1 f/f GC, and Foxo1 +/+ LZ B cells using genes differentially expressed (more than twofold) between Foxo1 +/+ DZ and Foxo1 +/+ LZ B cells (normalized log 2 values based on RNA-seq analysis). n = 3 biological replicates. (G) Gene set enrichment analysis showing the enrichment for genes up-regulated after ligation of CD40 (top) and BCR (bottom) compared of Foxo1 +/+ LZ B cells with Foxo1 f/f GC B cells. Error bars represent SD. Data are representative of three (B and C) or two independent experiments (D and E) and from one experiment (F and G). **, P
    Figure Legend Snippet: Foxo1 is required for GC maintenance. (A) Schematic illustration of the experimental protocol for B–D, F, and G. (B) Flow cytometry of NP-specific donor B cells (CD45.1 + B220 + NP + ). (C) Histograms representing the number of donor IgG1 − GC B cells (CD45.1 + B220 + NP + CD38 − IgG1 − ) and IgG1 + GC B cells (CD45.1 + B220 + NP + CD38 − IgG1 + ) in 10 6 splenocytes (left), and the ratio of DZ:LZ cells (right). n = 3 biological replicates. (D, left) DNA content measurement of Foxo1 +/+ and Foxo1 f/f LZ GC B cells assessed by 7-AAD staining. n = 5 and 3 biological replicates for tamoxifen and vehicle treatment, respectively. (right) Proliferation status of Foxo1 +/+ and Foxo1 f/f LZ GC B cells assessed by EdU incorporation 30 min after an EdU injection. n = 3 biological replicates. (E) Immunohistochemical analysis. (top) Schematic illustration of the experimental protocol. (bottom left) Representative images of immunofluorescence microscopy of spleen sections showing expression of CD45.1 ( Foxo1 f/f -derived donor cells), CD35 (FDC network), and IgD (follicular B cells). DZ and LZ defined by the presence of CD35 + FDCs are surrounded by dashed lines. Bars, 100 µm. (bottom right) Quantification of relative CD45.1 signal intensity in the DZ compared with that in the LZ. Each symbol represents a single GC, and red bars indicate the mean. n = 43 (tamoxifen) and 40 (vehicle) GC pooled from three animals. (F) Hierarchical clustering of the gene expression profiles of Foxo1 +/+ DZ, Foxo1 f/f GC, and Foxo1 +/+ LZ B cells using genes differentially expressed (more than twofold) between Foxo1 +/+ DZ and Foxo1 +/+ LZ B cells (normalized log 2 values based on RNA-seq analysis). n = 3 biological replicates. (G) Gene set enrichment analysis showing the enrichment for genes up-regulated after ligation of CD40 (top) and BCR (bottom) compared of Foxo1 +/+ LZ B cells with Foxo1 f/f GC B cells. Error bars represent SD. Data are representative of three (B and C) or two independent experiments (D and E) and from one experiment (F and G). **, P

    Techniques Used: Gas Chromatography, Flow Cytometry, Cytometry, Staining, Injection, Immunohistochemistry, Immunofluorescence, Microscopy, Expressing, Derivative Assay, RNA Sequencing Assay, Ligation

    5) Product Images from "Methods to Determine the Transcriptomes of Trypanosomes in Mixtures with Mammalian Cells: The Effects of Parasite Purification and Selective cDNA Amplification"

    Article Title: Methods to Determine the Transcriptomes of Trypanosomes in Mixtures with Mammalian Cells: The Effects of Parasite Purification and Selective cDNA Amplification

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0002806

    Amplification procedure. For explanation see text. A. RNA extraction; B. Mixing of trypanosome and HeLa RNA, if applicable; C. Location of cDNA primers; D. Location of SL reverse primer; E. Primers used for PCR; F. PCR product; G. Sheared PCR products; H. Sheared DNAs with Illumina adaptors; I. Sequence output; J. Sequences aligned to genome.
    Figure Legend Snippet: Amplification procedure. For explanation see text. A. RNA extraction; B. Mixing of trypanosome and HeLa RNA, if applicable; C. Location of cDNA primers; D. Location of SL reverse primer; E. Primers used for PCR; F. PCR product; G. Sheared PCR products; H. Sheared DNAs with Illumina adaptors; I. Sequence output; J. Sequences aligned to genome.

    Techniques Used: Amplification, RNA Extraction, Polymerase Chain Reaction, Sequencing

    6) Product Images from "Transcriptomic Analysis of Differentially Expressed Genes during Flower Organ Development in Genetic Male Sterile and Male Fertile Tagetes erecta by Digital Gene-Expression Profiling"

    Article Title: Transcriptomic Analysis of Differentially Expressed Genes during Flower Organ Development in Genetic Male Sterile and Male Fertile Tagetes erecta by Digital Gene-Expression Profiling

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0150892

    Linear regression analysis of the fold change of the gene expression ratios between DEG sequencing and qRT-PCR. 26 unigenes were selected for quantitative real-time PCR analysis to confirm the accuracy and reproducibility of the Illumina expression profiles using the same RNA samples that were used for DGE sequencing. The relative expression levels of the genes were calculated using the 2 −ΔΔCt method in qRT-PCR analysis. The DGE sequencing data were represented by the FPKM value of samples. Scatterplots were generated by the log 2 expression ratios from DGE sequencing data (x-axis) and qRT-PCR data (y-axis).
    Figure Legend Snippet: Linear regression analysis of the fold change of the gene expression ratios between DEG sequencing and qRT-PCR. 26 unigenes were selected for quantitative real-time PCR analysis to confirm the accuracy and reproducibility of the Illumina expression profiles using the same RNA samples that were used for DGE sequencing. The relative expression levels of the genes were calculated using the 2 −ΔΔCt method in qRT-PCR analysis. The DGE sequencing data were represented by the FPKM value of samples. Scatterplots were generated by the log 2 expression ratios from DGE sequencing data (x-axis) and qRT-PCR data (y-axis).

    Techniques Used: Expressing, Sequencing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Generated

    7) Product Images from "Finding Nemo: hybrid assembly with Oxford Nanopore and Illumina reads greatly improves the clownfish (Amphiprion ocellaris) genome assembly"

    Article Title: Finding Nemo: hybrid assembly with Oxford Nanopore and Illumina reads greatly improves the clownfish (Amphiprion ocellaris) genome assembly

    Journal: GigaScience

    doi: 10.1093/gigascience/gix137

    Mapping of MinION long reads, Illumina-assembled scaffolds, and RNA-sequencing reads of male and female A. ocellaris to the genomic region containing the cyp19a1a gene. Transcripts per million (TPM) values were calculated using Kallisto, version 0.43.1 .
    Figure Legend Snippet: Mapping of MinION long reads, Illumina-assembled scaffolds, and RNA-sequencing reads of male and female A. ocellaris to the genomic region containing the cyp19a1a gene. Transcripts per million (TPM) values were calculated using Kallisto, version 0.43.1 .

    Techniques Used: RNA Sequencing Assay

    8) Product Images from "Human NK cell development in hIL-7 and hIL-15 knockin NOD/SCID/IL2rgKO mice"

    Article Title: Human NK cell development in hIL-7 and hIL-15 knockin NOD/SCID/IL2rgKO mice

    Journal: Life Science Alliance

    doi: 10.26508/lsa.201800195

    Gene expression signature of NK cells in NSG hL-7xhIL-15 humanized mice. RNA sequencing was performed using RNA extracted from hCD56+ splenic NK cells of conventional NSG (NSG, n = 2) and hIL-7xhIL-15 KI NSG (hIL7xhIL-15 KI NSG, n = 6) humanized mice. (A) Differentially expressing genes are shown. Gene expression profiles of human NK cells of humanized mice were compared with those from NK cells recovered from human PBMCs. (B) Expression of genes related to cytokine and cytotoxicity are shown.
    Figure Legend Snippet: Gene expression signature of NK cells in NSG hL-7xhIL-15 humanized mice. RNA sequencing was performed using RNA extracted from hCD56+ splenic NK cells of conventional NSG (NSG, n = 2) and hIL-7xhIL-15 KI NSG (hIL7xhIL-15 KI NSG, n = 6) humanized mice. (A) Differentially expressing genes are shown. Gene expression profiles of human NK cells of humanized mice were compared with those from NK cells recovered from human PBMCs. (B) Expression of genes related to cytokine and cytotoxicity are shown.

    Techniques Used: Expressing, Mouse Assay, RNA Sequencing Assay

    9) Product Images from "The First Complete Genome Sequence of a Novel Tetrastichus brontispae RNA Virus-1 (TbRV-1)"

    Article Title: The First Complete Genome Sequence of a Novel Tetrastichus brontispae RNA Virus-1 (TbRV-1)

    Journal: Viruses

    doi: 10.3390/v11030257

    ( A ) Gel electrophoresis of viral RNA, and ( B ) the PCR products obtained from the viral cDNA and distilled water. M: Marker DL 2000 (TransGen Biotech); L1: PCR products with distilled water as template; L2: PCR products with cDNA template. The band was marked with a red triangle.
    Figure Legend Snippet: ( A ) Gel electrophoresis of viral RNA, and ( B ) the PCR products obtained from the viral cDNA and distilled water. M: Marker DL 2000 (TransGen Biotech); L1: PCR products with distilled water as template; L2: PCR products with cDNA template. The band was marked with a red triangle.

    Techniques Used: Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Marker

    10) Product Images from "Distinct Transcriptional and Alternative Splicing Signatures of Decidual CD4+ T Cells in Early Human Pregnancy"

    Article Title: Distinct Transcriptional and Alternative Splicing Signatures of Decidual CD4+ T Cells in Early Human Pregnancy

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.00682

    Human dCD4 T cells show a distinct transcriptional signature and upregulate genes related to immune system process as compared with autologous pCD4 T cells. Three healthy women at the first trimester of normal pregnancy were recruited and their dCD4 and pCD4 T cells were isolated by fluorescence-activated cell sorting (FACS). (A) Summary of mRNA-Seq data for the purified dCD4 and pCD4 T cells. RNA samples of paired dCD4 and pCD4 T cells from three individuals were sequenced on the Illumina Hiseq 2500 platform, yielding approximately 30–40 million 2 × 125-bp paired-end reads per sample, which were then mapped to the human reference genome (hg19 version). (B) Number and percentage of the differentially expressed genes ( P
    Figure Legend Snippet: Human dCD4 T cells show a distinct transcriptional signature and upregulate genes related to immune system process as compared with autologous pCD4 T cells. Three healthy women at the first trimester of normal pregnancy were recruited and their dCD4 and pCD4 T cells were isolated by fluorescence-activated cell sorting (FACS). (A) Summary of mRNA-Seq data for the purified dCD4 and pCD4 T cells. RNA samples of paired dCD4 and pCD4 T cells from three individuals were sequenced on the Illumina Hiseq 2500 platform, yielding approximately 30–40 million 2 × 125-bp paired-end reads per sample, which were then mapped to the human reference genome (hg19 version). (B) Number and percentage of the differentially expressed genes ( P

    Techniques Used: Isolation, Fluorescence, FACS, Purification

    11) Product Images from "Finding Nemo: hybrid assembly with Oxford Nanopore and Illumina reads greatly improves the clownfish (Amphiprion ocellaris) genome assembly"

    Article Title: Finding Nemo: hybrid assembly with Oxford Nanopore and Illumina reads greatly improves the clownfish (Amphiprion ocellaris) genome assembly

    Journal: GigaScience

    doi: 10.1093/gigascience/gix137

    Mapping of MinION long reads, Illumina-assembled scaffolds, and RNA-sequencing reads of male and female A. ocellaris to the genomic region containing the cyp19a1a gene. Transcripts per million (TPM) values were calculated using Kallisto, version 0.43.1 [ 46 ].
    Figure Legend Snippet: Mapping of MinION long reads, Illumina-assembled scaffolds, and RNA-sequencing reads of male and female A. ocellaris to the genomic region containing the cyp19a1a gene. Transcripts per million (TPM) values were calculated using Kallisto, version 0.43.1 [ 46 ].

    Techniques Used: RNA Sequencing Assay

    12) Product Images from "Distinct epigenetic programs regulate cardiac myocyte development and disease in the human heart in vivo"

    Article Title: Distinct epigenetic programs regulate cardiac myocyte development and disease in the human heart in vivo

    Journal: Nature Communications

    doi: 10.1038/s41467-017-02762-z

    Sorting and analysis of cardiac myocyte nuclei and representative epigenetic map. a Workflow for the isolation of human cardiac myocyte nuclei for epigenetic and transcriptomic analysis. b FACS analysis of nuclei isolated from adult non-failing left ventricular tissue (LV). Nuclei were stained with anti-pericentriolar material 1 (PCM1) and anti-phospholamban (PLN) antibodies to identify cardiac myocyte nuclei (red). c Proportion of cardiac myocyte nuclei in fetal (Fe, n = 8), infantile (I, n = 5), adult non-failing (NF, n = 5), and adult failing (F, n = 5) LV tissue (mean ± SEM). d Distribution of cardiac myocyte ploidy in fetal ( n = 8), infantile ( n = 5), non-failing ( n = 5), and failing ( n = 5) left ventricles (mean ± SEM). e Percentage of cardiac myocyte nuclei in LV tissue before sorting (pre, open columns) and cardiac myocyte nuclei purity after FACS sorting (post, n = 3, mean ± SEM). f Original traces of RNA expression, mCpG, and histone marks of the troponin I type 1 ( TNNI1 ) and troponin T type 2 ( TNNT2 ) gene region. CpG islands, low methylated regions (LMR), unmethylated regions (UMR), genic unmethylated regions (gUMR), and the chromatin state are annotated. Gray areas highlight differentially CpG-methylated regions and genes. Shown are data from n biological replicates: mCpG, n = 3–5; H3K27ac, H3K9ac, H3K36me3, H3K4me1, H3K4me3, H3K27me3, and H3K9me3, n = 3; RNA, n = 3–4; * vs. Fe, p
    Figure Legend Snippet: Sorting and analysis of cardiac myocyte nuclei and representative epigenetic map. a Workflow for the isolation of human cardiac myocyte nuclei for epigenetic and transcriptomic analysis. b FACS analysis of nuclei isolated from adult non-failing left ventricular tissue (LV). Nuclei were stained with anti-pericentriolar material 1 (PCM1) and anti-phospholamban (PLN) antibodies to identify cardiac myocyte nuclei (red). c Proportion of cardiac myocyte nuclei in fetal (Fe, n = 8), infantile (I, n = 5), adult non-failing (NF, n = 5), and adult failing (F, n = 5) LV tissue (mean ± SEM). d Distribution of cardiac myocyte ploidy in fetal ( n = 8), infantile ( n = 5), non-failing ( n = 5), and failing ( n = 5) left ventricles (mean ± SEM). e Percentage of cardiac myocyte nuclei in LV tissue before sorting (pre, open columns) and cardiac myocyte nuclei purity after FACS sorting (post, n = 3, mean ± SEM). f Original traces of RNA expression, mCpG, and histone marks of the troponin I type 1 ( TNNI1 ) and troponin T type 2 ( TNNT2 ) gene region. CpG islands, low methylated regions (LMR), unmethylated regions (UMR), genic unmethylated regions (gUMR), and the chromatin state are annotated. Gray areas highlight differentially CpG-methylated regions and genes. Shown are data from n biological replicates: mCpG, n = 3–5; H3K27ac, H3K9ac, H3K36me3, H3K4me1, H3K4me3, H3K27me3, and H3K9me3, n = 3; RNA, n = 3–4; * vs. Fe, p

    Techniques Used: Isolation, FACS, Staining, RNA Expression, Methylation

    13) Product Images from "Integrated analysis of hepatic mRNA and miRNA profiles identified molecular networks and potential biomarkers of NAFLD"

    Article Title: Integrated analysis of hepatic mRNA and miRNA profiles identified molecular networks and potential biomarkers of NAFLD

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-25743-8

    Real-time PCR validation of several representative expressed mRNAs and miRNAs. The x-axis represents RNA names, and the y-axis represents log 2 (fold change) based on the ratios between the NAFLD and normal groups’ average expression values. Blue bars represent data yielded by real-time qPCR, and red points represent data obtained by RNA sequencing.
    Figure Legend Snippet: Real-time PCR validation of several representative expressed mRNAs and miRNAs. The x-axis represents RNA names, and the y-axis represents log 2 (fold change) based on the ratios between the NAFLD and normal groups’ average expression values. Blue bars represent data yielded by real-time qPCR, and red points represent data obtained by RNA sequencing.

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, RNA Sequencing Assay

    Hierarchical cluster of representative mRNA and miRNA expression across biological replicate samples. ( A ) Heatmap of representative mRNAs. ( B ) Heatmap of representative miRNAs. RNA expression level is represented by colors, with bright blue indicating high values and bright yellow indicating low values.
    Figure Legend Snippet: Hierarchical cluster of representative mRNA and miRNA expression across biological replicate samples. ( A ) Heatmap of representative mRNAs. ( B ) Heatmap of representative miRNAs. RNA expression level is represented by colors, with bright blue indicating high values and bright yellow indicating low values.

    Techniques Used: Expressing, RNA Expression

    14) Product Images from "Integrated microRNA and mRNA analysis in the pathogenic filamentous fungus Trichophyton rubrum"

    Article Title: Integrated microRNA and mRNA analysis in the pathogenic filamentous fungus Trichophyton rubrum

    Journal: BMC Genomics

    doi: 10.1186/s12864-018-5316-3

    Validation of RNA-Seq results by qRT-PCR. Three biological replicates were performed. * indicates significant difference of milRNA/mRNA expression level in conidial vs. mycelial stages (*: P
    Figure Legend Snippet: Validation of RNA-Seq results by qRT-PCR. Three biological replicates were performed. * indicates significant difference of milRNA/mRNA expression level in conidial vs. mycelial stages (*: P

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

    15) Product Images from "SUMOylation of the m6A-RNA methyltransferase METTL3 modulates its function"

    Article Title: SUMOylation of the m6A-RNA methyltransferase METTL3 modulates its function

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky156

    SUMO1 modification of METTL3 represses its RNA m 6 A methyltransferase activity. (A–E) Polyadenylated mRNAs were purified for the dot-blot assay (upper panels), and cell lysates were used for immunoblotting with indicated antibodies (lower panels). ( A ) METTL3 is a main component responsible for the abundance of m 6 A in mRNAs. The abundance of m 6 A in mRNAs from shControl or shMETTL3 293T and H1299 cells was detected by the Dot-blot assay with anti-m 6 A antibody, and equal loading of the mRNAs was verified by methylene blue staining (upper panels). METTL3 knockdown efficiency in 293T and H1299 cells was shown (lower panels). ( B ) The level of m 6 A in mRNAs is low in the high SUMOylation status in SENP1 knockdown cells. ( C ) SUMOylation of METTL3 reduces its m 6 A methyltransferase activity. HA-METTL3 with or without His-SUMO1/Flag-Ubc9 were transfected into 293T cells. (D–F) The SUMO-site mutataion 4KR (K 177/211/212/215 R) of METTL3 significantly enhances its m 6 A methyltransferase activity. ( D ) HA-METTL3-WT or -4KR was transiently transfeced into 293T cells, and ( E ) HA-METTL3-WT or -4KR was stably re-expressed H1299-shMETTL3 by using the lentiviral system. ( F ) HA-METTL3-WT or -4KR were transfected with or without His-SUMO1/Flag-Ubc9 into 293T cells. The SUMOylation assays and dot-blot assays were performed as described before. ( G ) LC–MS/MS quantification of the m 6 A/A ratio in polyadenylated RNAs purified from H1299-shMETTL3 cells with METTL3-WT or METTL3-4KR. Error bars indicate mean ± S.D. (two technical replicates). ( H ) The in vitro RNA N6-adenosine methylation activity was tested using purified Flag-METTL3-WT, SUMOlated Flag-METTL3-WT or Flag-METTL3-4KR proteins in combination with purified Flag-METTL14 and RNA-probe (Seq1) with consensus sequence of ‘GGACU’. The methylation of RNA-probe was measured by immunoblotting with the m 6 A antibody.
    Figure Legend Snippet: SUMO1 modification of METTL3 represses its RNA m 6 A methyltransferase activity. (A–E) Polyadenylated mRNAs were purified for the dot-blot assay (upper panels), and cell lysates were used for immunoblotting with indicated antibodies (lower panels). ( A ) METTL3 is a main component responsible for the abundance of m 6 A in mRNAs. The abundance of m 6 A in mRNAs from shControl or shMETTL3 293T and H1299 cells was detected by the Dot-blot assay with anti-m 6 A antibody, and equal loading of the mRNAs was verified by methylene blue staining (upper panels). METTL3 knockdown efficiency in 293T and H1299 cells was shown (lower panels). ( B ) The level of m 6 A in mRNAs is low in the high SUMOylation status in SENP1 knockdown cells. ( C ) SUMOylation of METTL3 reduces its m 6 A methyltransferase activity. HA-METTL3 with or without His-SUMO1/Flag-Ubc9 were transfected into 293T cells. (D–F) The SUMO-site mutataion 4KR (K 177/211/212/215 R) of METTL3 significantly enhances its m 6 A methyltransferase activity. ( D ) HA-METTL3-WT or -4KR was transiently transfeced into 293T cells, and ( E ) HA-METTL3-WT or -4KR was stably re-expressed H1299-shMETTL3 by using the lentiviral system. ( F ) HA-METTL3-WT or -4KR were transfected with or without His-SUMO1/Flag-Ubc9 into 293T cells. The SUMOylation assays and dot-blot assays were performed as described before. ( G ) LC–MS/MS quantification of the m 6 A/A ratio in polyadenylated RNAs purified from H1299-shMETTL3 cells with METTL3-WT or METTL3-4KR. Error bars indicate mean ± S.D. (two technical replicates). ( H ) The in vitro RNA N6-adenosine methylation activity was tested using purified Flag-METTL3-WT, SUMOlated Flag-METTL3-WT or Flag-METTL3-4KR proteins in combination with purified Flag-METTL14 and RNA-probe (Seq1) with consensus sequence of ‘GGACU’. The methylation of RNA-probe was measured by immunoblotting with the m 6 A antibody.

    Techniques Used: Modification, Activity Assay, Purification, Dot Blot, Staining, Hemagglutination Assay, Transfection, Stable Transfection, Liquid Chromatography, Mass Spectrometry, In Vitro, Methylation, Sequencing

    SUMOylation of METTL3 down-regulates m 6 A modification in mRNAs resulting in the alternation of gene expression profile. ( A ) Cumulative distribution curve for the abundance of m 6 A modification across the transcriptome of H1299-shMETTL3 cells re-expressing METTL3-WT or METTL3-4KR. ( B ) Distribution of m 6 A peaks across around stop codons and 3′ UTRs of the entire set of mRNA transcripts. ( C ) Comparison of the abundance of m 6 A peaks across the transcriptome of H1299-shMETTL3 cells re-expressing METTL3-WT or METTL3-4KR. The fold-change ≥2.0 was considered to be significant, which was the abundance of m 6 A peaks of METTL3-4KR relative to METTL3-WT. IP/Input, was referred to as the abundance of m 6 A peak in mRNAs detected in MeRIP m 6 A-Seq (IP) normalized by that detected in RNA-Seq (Input). ( D ) Heatmap showing the alternation of mRNA expression profiles in H1299-shMETTL3 cells re-expressing METTL3-WT or METTL3-4KR.
    Figure Legend Snippet: SUMOylation of METTL3 down-regulates m 6 A modification in mRNAs resulting in the alternation of gene expression profile. ( A ) Cumulative distribution curve for the abundance of m 6 A modification across the transcriptome of H1299-shMETTL3 cells re-expressing METTL3-WT or METTL3-4KR. ( B ) Distribution of m 6 A peaks across around stop codons and 3′ UTRs of the entire set of mRNA transcripts. ( C ) Comparison of the abundance of m 6 A peaks across the transcriptome of H1299-shMETTL3 cells re-expressing METTL3-WT or METTL3-4KR. The fold-change ≥2.0 was considered to be significant, which was the abundance of m 6 A peaks of METTL3-4KR relative to METTL3-WT. IP/Input, was referred to as the abundance of m 6 A peak in mRNAs detected in MeRIP m 6 A-Seq (IP) normalized by that detected in RNA-Seq (Input). ( D ) Heatmap showing the alternation of mRNA expression profiles in H1299-shMETTL3 cells re-expressing METTL3-WT or METTL3-4KR.

    Techniques Used: Modification, Expressing, RNA Sequencing Assay

    16) Product Images from "BMPs as new insulin sensitizers: enhanced glucose uptake in mature 3T3-L1 adipocytes via PPARγ and GLUT4 upregulation"

    Article Title: BMPs as new insulin sensitizers: enhanced glucose uptake in mature 3T3-L1 adipocytes via PPARγ and GLUT4 upregulation

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-17595-5

    BMP2 and BMP6 stimulation regulate mRNA levels of metabolic enzymes/transporters and visfatin. Validation of genes responding to BMP stimulation by qRT-PCR. Adipocytes were treated as outlined in Fig. 2 . Data are presented as means + SEM of two independent experiments different from samples used in the RNAseq experiment, n = 2 for a-c,e-i; n = 3 for d. Lpl ( a ) and Fasn ( b ) were selected from Cluster A. Plin1 ( c ), Id1 ( e ) and Rxra ( f ) and Nampt ( g ) coding for Pre-B-cell colony-enhancing factor 1 (PBEF1) or visfatin were selected from Cluster B. Glut-1 ( d ) was not found to be significantly regulated in the RNA-Seq approach, but selected for validation as it represents the second glucose transporter present in adipocytes. Fatp1 ( h ) and Lep ( i ) were selected from Cluster D. Asteriks denote PPARγ target genes.
    Figure Legend Snippet: BMP2 and BMP6 stimulation regulate mRNA levels of metabolic enzymes/transporters and visfatin. Validation of genes responding to BMP stimulation by qRT-PCR. Adipocytes were treated as outlined in Fig. 2 . Data are presented as means + SEM of two independent experiments different from samples used in the RNAseq experiment, n = 2 for a-c,e-i; n = 3 for d. Lpl ( a ) and Fasn ( b ) were selected from Cluster A. Plin1 ( c ), Id1 ( e ) and Rxra ( f ) and Nampt ( g ) coding for Pre-B-cell colony-enhancing factor 1 (PBEF1) or visfatin were selected from Cluster B. Glut-1 ( d ) was not found to be significantly regulated in the RNA-Seq approach, but selected for validation as it represents the second glucose transporter present in adipocytes. Fatp1 ( h ) and Lep ( i ) were selected from Cluster D. Asteriks denote PPARγ target genes.

    Techniques Used: Quantitative RT-PCR, RNA Sequencing Assay

    17) Product Images from "Effects of anticholinergic agent on miRNA profiles and transcriptomes in a murine model of allergic rhinitis"

    Article Title: Effects of anticholinergic agent on miRNA profiles and transcriptomes in a murine model of allergic rhinitis

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2017.7411

    Venn diagram of differentially expressed (A) mRNAs and (B) miRNAs. Control: Group A (n=7) and group B (n=8). Model: Group C (n=8), group D (n=10) and group E (n=9). When mRNAs or miRNAs exhibit similar characteristics, they appear in the overlapping boxes. mRNA, messenger RNA; miRNA, microRNA.
    Figure Legend Snippet: Venn diagram of differentially expressed (A) mRNAs and (B) miRNAs. Control: Group A (n=7) and group B (n=8). Model: Group C (n=8), group D (n=10) and group E (n=9). When mRNAs or miRNAs exhibit similar characteristics, they appear in the overlapping boxes. mRNA, messenger RNA; miRNA, microRNA.

    Techniques Used:

    18) Product Images from "Polycomb- and Methylation-Independent Roles of EZH2 as a Transcription Activator"

    Article Title: Polycomb- and Methylation-Independent Roles of EZH2 as a Transcription Activator

    Journal: Cell reports

    doi: 10.1016/j.celrep.2018.11.035

    EZH2 Directly Activates AR Gene Transcription (A) EZH2 protein occupies the AR gene promoter. EZH2 ChIP-seq was performed in LNCaP cells with an antibody targeting endogenous EZH2 (top). HA ChIP-seq was performed using an anti-HA antibody in LNCaP cells with ectopic HA-EZH2 overexpression. Two biological replicates are shown (center and bottom). (B) ChIP-qPCR showing EZH2 binding along the AR gene promoter. ChIP was performed in LNCaP cells using anti-EZH2 and IgG antibodies and then subjected to qPCR using primer pairs targeting ~60-bp sliding windows within −1 kb to +3 kb of the AR gene. The x axis indicates the central location of the PCR products relative to the AR TSS. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (C) Different regions (of 400 bp) of the AR promoter (from 0 to +3 kb) were cloned into the pRetroX-Tight-Pur-Luc vector and transfected into 293T cells, which were then subjected to ChIP by anti-EZH2 or IgG. EZH2 occupancy at the ectopically expressed AR promoter was determined by qPCR using a common forward primer targeting the vector sequence and a reverse primer specific to each fragment. Data shown are mean (±SEM) of technical replicates from one representative experiment of two. (D) Various AR promoter regions were cloned into the pGL4.10 vector and transfected into 293T cells with either control pLVX or HA-EZH2 overexpression. Cells were then subjected to luciferase reporter assays. Results were normalized to the Renilla internal control. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (E) Schematic view of the AR promoter sequence starting from the transcription start site (TSS). The sgRNAs were labeled sgAR1 to 4, their sequences are shown in green font, and their distances to the AR TSS are marked as numbers. The primers (F2 and R2) for PCR validation are shown in purple. (F and G) The distal AR promoter region is required for EZH2 activation of AR transcription. LNCaP cells were infected with lentiCRISPR-Cas9 containing the pLENTI.V2 control, sgAR1+2, sgAR3+4, or sgAR1+4 for 48 hr. CRISPR-Cas9-mediated genome editing was confirmed by Sanger sequencing (F) and genomic DNA PCR (G) using primers F2 and R2 (indicated in A and E). (H) CRISPR-Cas9-edited LNCaP cells were transfected with control or EZH2-targeting siRNA for 48 hr. Total RNA was harvested and subjected to RT-PCR analysis using F2 and R2, which are expected to yield a wild-type (AR WT, top band with black asterisk) and a CRISPR-Cas9-deleted (AR del, bottom bands with red asterisk) AR mRNA.
    Figure Legend Snippet: EZH2 Directly Activates AR Gene Transcription (A) EZH2 protein occupies the AR gene promoter. EZH2 ChIP-seq was performed in LNCaP cells with an antibody targeting endogenous EZH2 (top). HA ChIP-seq was performed using an anti-HA antibody in LNCaP cells with ectopic HA-EZH2 overexpression. Two biological replicates are shown (center and bottom). (B) ChIP-qPCR showing EZH2 binding along the AR gene promoter. ChIP was performed in LNCaP cells using anti-EZH2 and IgG antibodies and then subjected to qPCR using primer pairs targeting ~60-bp sliding windows within −1 kb to +3 kb of the AR gene. The x axis indicates the central location of the PCR products relative to the AR TSS. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (C) Different regions (of 400 bp) of the AR promoter (from 0 to +3 kb) were cloned into the pRetroX-Tight-Pur-Luc vector and transfected into 293T cells, which were then subjected to ChIP by anti-EZH2 or IgG. EZH2 occupancy at the ectopically expressed AR promoter was determined by qPCR using a common forward primer targeting the vector sequence and a reverse primer specific to each fragment. Data shown are mean (±SEM) of technical replicates from one representative experiment of two. (D) Various AR promoter regions were cloned into the pGL4.10 vector and transfected into 293T cells with either control pLVX or HA-EZH2 overexpression. Cells were then subjected to luciferase reporter assays. Results were normalized to the Renilla internal control. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (E) Schematic view of the AR promoter sequence starting from the transcription start site (TSS). The sgRNAs were labeled sgAR1 to 4, their sequences are shown in green font, and their distances to the AR TSS are marked as numbers. The primers (F2 and R2) for PCR validation are shown in purple. (F and G) The distal AR promoter region is required for EZH2 activation of AR transcription. LNCaP cells were infected with lentiCRISPR-Cas9 containing the pLENTI.V2 control, sgAR1+2, sgAR3+4, or sgAR1+4 for 48 hr. CRISPR-Cas9-mediated genome editing was confirmed by Sanger sequencing (F) and genomic DNA PCR (G) using primers F2 and R2 (indicated in A and E). (H) CRISPR-Cas9-edited LNCaP cells were transfected with control or EZH2-targeting siRNA for 48 hr. Total RNA was harvested and subjected to RT-PCR analysis using F2 and R2, which are expected to yield a wild-type (AR WT, top band with black asterisk) and a CRISPR-Cas9-deleted (AR del, bottom bands with red asterisk) AR mRNA.

    Techniques Used: Chromatin Immunoprecipitation, Over Expression, Real-time Polymerase Chain Reaction, Binding Assay, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Transfection, Sequencing, Luciferase, Labeling, Activation Assay, Infection, CRISPR, Reverse Transcription Polymerase Chain Reaction

    19) Product Images from "The primary transcriptome, small RNAs and regulation of antimicrobial resistance in Acinetobacter baumannii ATCC 17978"

    Article Title: The primary transcriptome, small RNAs and regulation of antimicrobial resistance in Acinetobacter baumannii ATCC 17978

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky603

    sRNA in A. baumannii ATCC 17978. ( A ) Normalized, mapped sequence reads from RNA-seq show the expression of sRNAs 17, 37, 75, 76, 77, 84, 99 and 100 (yellow arrows). Curved arrows depict TSS identified in this study and lollipop structures are predicted rho-independent terminators. Northern blotting of selected sRNAs are shown to the right. RNA was isolated from ESP and five μg of total RNA was loaded per lane. The sRNA sizes below the individual blots have been predicted from dRNA-seq data. ( B ) Sequence alignment of Group I and Group III sRNAs created with the Geneious Software (v. 8.1.8); colored bases indicate conservation in at least 50% of aligned sequences (A, red; C, blue; G, yellow; T, green). The riboprobes used in Northern blotting are depicted as black bars atop the sRNA alignments.
    Figure Legend Snippet: sRNA in A. baumannii ATCC 17978. ( A ) Normalized, mapped sequence reads from RNA-seq show the expression of sRNAs 17, 37, 75, 76, 77, 84, 99 and 100 (yellow arrows). Curved arrows depict TSS identified in this study and lollipop structures are predicted rho-independent terminators. Northern blotting of selected sRNAs are shown to the right. RNA was isolated from ESP and five μg of total RNA was loaded per lane. The sRNA sizes below the individual blots have been predicted from dRNA-seq data. ( B ) Sequence alignment of Group I and Group III sRNAs created with the Geneious Software (v. 8.1.8); colored bases indicate conservation in at least 50% of aligned sequences (A, red; C, blue; G, yellow; T, green). The riboprobes used in Northern blotting are depicted as black bars atop the sRNA alignments.

    Techniques Used: Sequencing, RNA Sequencing Assay, Expressing, Northern Blot, Isolation, End-sequence Profiling, Software

    20) Product Images from "Enhanced Anti-lymphoma Activity of CAR19-iNKT Cells Underpinned by Dual CD19 and CD1d Targeting"

    Article Title: Enhanced Anti-lymphoma Activity of CAR19-iNKT Cells Underpinned by Dual CD19 and CD1d Targeting

    Journal: Cancer Cell

    doi: 10.1016/j.ccell.2018.08.017

    Transcriptional and Epigenetic Basis for Enhancing CAR19-iNKT Cell Reactivity (A) CD1D mRNA quantification by qPCR in CLL cells from two patients upon ATRA treatment (10 −6 M) for 0–96 hr. (B and C) Bar charts (B) and flow cytometry histograms (C) showing CD1d expression on malignant B cells upon ATRA treatment and mean fluorescent intensity (MFI) analysis of CD1d expression in comparison with isotype control. (D) Cytotoxicity of second- and third-generation CAR19-T and -NKT cells against αGalCer-pulsed CLL cells pre-treated with 0.1% DMSO control or 10 −6 M ATRA. Error bars represent SEM of triplicate assays. (E) ChIP-qPCR assay for H3K4me3 and H3K27me3 enrichment in the promoter of CD1D using IgG as control in U266 cells. GAPDH is an active gene control, while HOXA2 is a repressed gene control. ChIP data are shown as a percentage of the input chromatin. (F) ChIP-re-ChIP qPCR assay showing fold enrichment of H3K27me3 or IgG control after immunoprecipitation (IP) against H3K4me3. (G) ChIP-qPCR assay against RNA Pol II for Ser5 over Ser2 phosphorylated form at the promoter of the indicated genes. (H) ChIP-qPCR assay against RARα, EZH2, and Ig control at the promoters of the genes shown. (I) ChIP-re-ChIP qPCR assay showing enrichment of EZH2 or IgG control after IP against RARα in U266 cells for –(I) (n = 3). (J) qPCR quantification of CD1D mRNA in U266 cells treated with 0.1% DMSO, 10 −6 M GSK343, 10 −6 M ATRA or 10 −6 M GSK343 plus 10 −6 M ATRA. Values are normalized to CD1D mRNA expression levels in normal peripheral PB B cells (n = 3). ND, not detectable. (K and L) Relative MFI analysis (K) and histogram depiction (L) of CD1d expression in comparison with isotype control in U266 cells from the same experiment shown in (J). Error bars represent SEM. See also Figure S4 .
    Figure Legend Snippet: Transcriptional and Epigenetic Basis for Enhancing CAR19-iNKT Cell Reactivity (A) CD1D mRNA quantification by qPCR in CLL cells from two patients upon ATRA treatment (10 −6 M) for 0–96 hr. (B and C) Bar charts (B) and flow cytometry histograms (C) showing CD1d expression on malignant B cells upon ATRA treatment and mean fluorescent intensity (MFI) analysis of CD1d expression in comparison with isotype control. (D) Cytotoxicity of second- and third-generation CAR19-T and -NKT cells against αGalCer-pulsed CLL cells pre-treated with 0.1% DMSO control or 10 −6 M ATRA. Error bars represent SEM of triplicate assays. (E) ChIP-qPCR assay for H3K4me3 and H3K27me3 enrichment in the promoter of CD1D using IgG as control in U266 cells. GAPDH is an active gene control, while HOXA2 is a repressed gene control. ChIP data are shown as a percentage of the input chromatin. (F) ChIP-re-ChIP qPCR assay showing fold enrichment of H3K27me3 or IgG control after immunoprecipitation (IP) against H3K4me3. (G) ChIP-qPCR assay against RNA Pol II for Ser5 over Ser2 phosphorylated form at the promoter of the indicated genes. (H) ChIP-qPCR assay against RARα, EZH2, and Ig control at the promoters of the genes shown. (I) ChIP-re-ChIP qPCR assay showing enrichment of EZH2 or IgG control after IP against RARα in U266 cells for –(I) (n = 3). (J) qPCR quantification of CD1D mRNA in U266 cells treated with 0.1% DMSO, 10 −6 M GSK343, 10 −6 M ATRA or 10 −6 M GSK343 plus 10 −6 M ATRA. Values are normalized to CD1D mRNA expression levels in normal peripheral PB B cells (n = 3). ND, not detectable. (K and L) Relative MFI analysis (K) and histogram depiction (L) of CD1d expression in comparison with isotype control in U266 cells from the same experiment shown in (J). Error bars represent SEM. See also Figure S4 .

    Techniques Used: Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Expressing, Chromatin Immunoprecipitation, Immunoprecipitation

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    RNA Sequencing Assay:

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    Article Snippet: Paragraph title: Library preparation and RNA-seq ... The cDNA library was prepared using a NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs) following the manufacturer’s instructions.

    Article Title: Small non-coding RNA landscape of extracellular vesicles from human stem cells
    Article Snippet: Paragraph title: Small RNA sequencing ... NGS libraries were prepared using the NEBNext® Small RNA Library preparation kit (New England Biolabs), consisting of adapter ligation, cDNA conversion, PCR amplification (18 cycles) and purification.

    Article Title: Transforming growth factor beta1 targets estrogen receptor signaling in bronchial epithelial cells
    Article Snippet: Paragraph title: RNA-Seq library preparation and sequencing ... 2 μL of 1:2000 diluted External RNA Controls Consortium (ERCC) Spike-In Mix (Ambion™ 4,456,740, ThermoFisher Scientific Inc.) was added to 100 ng of high-quality total RNA followed by mRNA isolation using NEBNext Poly(A) mRNA Magnetic Isolation module (New England Biolabs E7490, Ipswich, MA) and RNA library construction with NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs E7530) according to the manufacturer’s user guide.

    Article Title: In Utero Heat Stress Alters the Offspring Epigenome
    Article Snippet: RNA with 28S/18S > 1 and an RNA integrity number ≥7 were used for library construction. .. RNA-Seq library was constructed using NEBNext® Ultra™ RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA) following manufacturer’s recommendations. .. Libraries were sequenced on the Illumina HiSeq.

    Article Title: Sea cucumber genome provides insights into saponin biosynthesis and aestivation regulation
    Article Snippet: Total mRNA was extracted from each sample ( > 1000 embryos/larvae per developmental stage) by following the protocol described by Du et al. . .. All RNA-seq libraries were constructed using the NEB Next mRNA Library Prep Kit by following the manufacturer’s instructions and then were subjected to paired-end 100-bp (PE100) sequencing on the Illumina HiSeq 2000 platform. .. Sequencing reads were aligned to the A . japonicus genome using STAR aligner with its default parameters.

    Article Title: Functional Insights into the Roles of Hormones in the Dendrobium officinale-Tulasnella sp. Germinated Seed Symbiotic Association
    Article Snippet: For each sample, 3 µg of the total RNA was used as input material for the RNA-Seq library preparations. .. Sequencing libraries were generated using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, Ipswitch, MA, USA) following the manufacturer’s recommendations, and index codes were added to attribute the sequencing reads to each sample.

    Article Title: Characterization of human plasma-derived exosomal RNAs by deep sequencing
    Article Snippet: To accomplish this goal, we selected plasma samples (samples A, B and C) from three anonymous blood donors and split each sample into two for technical replication. .. We tested the six samples (A1 and A2, B1 and B2, C1 and C2) using two small RNA library preparation kits: NEBNext multiplex small RNA library preparation kit (NEB, New England Biolab, Ipswich, MA, USA) and NEXTflex small RNA sequencing kit (Bioo Scientific, Austin, TX, USA). .. We also tested samples A1 and A2 using the TruSeq small RNA sample preparation kit (Illumina, San Diego, CA, USA).

    Article Title: Transcriptomic variation of hepatopancreas reveals the energy metabolism and biological processes associated with molting in Chinese mitten crab, Eriocheir sinensis
    Article Snippet: The RNA integrity and quantity were determined using an Agilent 2100 Bioanalyzer (Agilent, Shanghai, China). .. A total of 4 μg RNA with RNA integrity number above 7.0 was used for RNA-Seq library construction using the NEBNext® UltraTM RNA Library Prep Kit for Illumina (NEB, USA). .. Indexed libraries were then pooled and sequenced on Illumina HiseqTM 2500, with 150 bp pair-end reads produced.

    Article Title: Global analyses of endonucleolytic cleavage in mammals reveal expanded repertoires of cleavage-inducing small RNAs and their targets
    Article Snippet: Spike-in RNAs (Exiqon) were added into 1 μg total RNA before library construction. .. Small RNA sequencing libraries were generated using NEBNext Small RNA library Prep kit and NEBNext multiplex oligos for Illumina according to the manufacturer's instructions (NEB). .. The final libraries were purified from 6% PAGE gel, and their concentrations were measured using Qubit fluorometric assay (Life Technologies).

    Fluorescence:

    Article Title: Potent and Targeted Activation of Latent HIV-1 Using the CRISPR/dCas9 Activator Complex
    Article Snippet: To control for transfection efficiency, cells were sorted by fluorescence activated cell sorting based on GFP expression using the BD FACSAria II 72 hours posttransfection. .. The sequencing library was prepared using the NEBNext Ultra RNA Library Prep Kit reagent.

    Magnetic Beads:

    Article Title: Comparative transcriptomics identifies genes differentially expressed in the intestine of a new fast-growing strain of common carp with higher unsaturated fatty acid content in muscle
    Article Snippet: RNA integrity and concentration were assessed using an Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). cDNA libraries were generated using NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, USA) following the manufacturer’s recommendations. .. RNA integrity and concentration were assessed using an Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). cDNA libraries were generated using NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, USA) following the manufacturer’s recommendations.

    Isolation:

    Article Title: Small non-coding RNA landscape of extracellular vesicles from human stem cells
    Article Snippet: Good performance of Exiqons EV isolation kit has been reported . .. NGS libraries were prepared using the NEBNext® Small RNA Library preparation kit (New England Biolabs), consisting of adapter ligation, cDNA conversion, PCR amplification (18 cycles) and purification.

    Article Title: Whole body transcriptomes and new insights into the biology of the tick Ixodes ricinus
    Article Snippet: The library preparation kit was NEBNext® Ultra Directional RNA Library Prep Kit, NEB Art. .. The library preparation kit was NEBNext® Ultra Directional RNA Library Prep Kit, NEB Art.

    Article Title: Transforming growth factor beta1 targets estrogen receptor signaling in bronchial epithelial cells
    Article Snippet: The RINs of all total RNA samples were 9.7–10. .. 2 μL of 1:2000 diluted External RNA Controls Consortium (ERCC) Spike-In Mix (Ambion™ 4,456,740, ThermoFisher Scientific Inc.) was added to 100 ng of high-quality total RNA followed by mRNA isolation using NEBNext Poly(A) mRNA Magnetic Isolation module (New England Biolabs E7490, Ipswich, MA) and RNA library construction with NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs E7530) according to the manufacturer’s user guide. .. Specifically, 100 ng of total RNA together with 2 μL of 1:2000 diluted ERCC was added to extracted mRNA with 15 μL of NEBNext Magnetic Oligo d(T)25 and fragmented in NEBNext First Strand Synthesis Buffer by heating at 94 °C for 8 min, followed by first-strand cDNA synthesis using reverse transcriptase and random primers.

    Article Title: In Utero Heat Stress Alters the Offspring Epigenome
    Article Snippet: Paragraph title: RNA isolation and RNA-sequencing ... RNA-Seq library was constructed using NEBNext® Ultra™ RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA) following manufacturer’s recommendations.

    Article Title: Comparative transcriptomics identifies genes differentially expressed in the intestine of a new fast-growing strain of common carp with higher unsaturated fatty acid content in muscle
    Article Snippet: Paragraph title: RNA isolation, cDNA library construction and Illumina sequencing ... RNA integrity and concentration were assessed using an Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). cDNA libraries were generated using NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, USA) following the manufacturer’s recommendations.

    Article Title: Transcriptomic variation of hepatopancreas reveals the energy metabolism and biological processes associated with molting in Chinese mitten crab, Eriocheir sinensis
    Article Snippet: Paragraph title: RNA isolation and RNA-Seq library preparation ... A total of 4 μg RNA with RNA integrity number above 7.0 was used for RNA-Seq library construction using the NEBNext® UltraTM RNA Library Prep Kit for Illumina (NEB, USA).

    Article Title: Transcriptome-Wide Analysis of Hepatitis B Virus-Mediated Changes to Normal Hepatocyte Gene Expression
    Article Snippet: For the secondary dataset, total RNA was isolated using the mirVana RNA isolation kit from triplicate samples of PRHs; these PRHs were isolated from a separate rat liver and were infected with different preparations of AdGFP and AdGFP-HBV than were used to generate the primary dataset. .. Total RNA was divided for use in downstream miRNA analyses (see below) or for quality control, polyA selection, cDNA library preparation, and the first round of sequencing by Genewiz, Inc (South Plainfield, NJ). cDNA library preparation was done using the NEBNext Ultra RNA Library Preparation Kit (New England Biolabs, Ipswich, MA), and the first round of sequencing was done using the Illumina HiSeq2500 platform to generate 1 x 50bp reads.

    Article Title: Whole transcriptome profiling reveals the RNA content of motor axons
    Article Snippet: Libraries were pooled and purified with AMPure XP beads for sequencing. .. Three replicate total RNAseq libraries from 5 ng HBRR input each were prepared using the NEBNext Ultra RNA Library Kit for Illumina (NEB) with omission of the mRNA isolation step. .. Instead, fragmentation was performed directly on total RNA by adding 4 μl of first strand reaction buffer (5×) and 1 μl random primers to 5 μl of the 1 ng/μl HBRR/ERCC mix (see above) and heating the mixture at 94°C for 15 min.

    Article Title: Mating-Induced Transcriptome Changes in the Reproductive Tract of Female Aedes aegypti
    Article Snippet: The concentration of RNA in each sample was measured using a Qubit spectrophotometer (Invitrogen, Grand Island, NY), and the quality of RNA was measured on a Fragment Analyzer (Advanced Analytical Technologies, Inc., Ames, IA). .. From total RNA, mRNA was extracted using the poly-A mRNA Magnetic Isolation Module (New England Biolabs, Inc., Ipswich, MA). cDNA libraries for each replicate were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs, Inc., Ipswich, MA). .. NEBNext Multiplex Oligos for Illumina (New England Biolabs, Inc., Ipswich, MA) were ligated to each library prior to sequencing.

    Article Title: Potent and Targeted Activation of Latent HIV-1 Using the CRISPR/dCas9 Activator Complex
    Article Snippet: Total RNA was isolated from ~25,000–30,000 sorted cells using the Promega Maxwell 16 LEV Simply RNA Cell Kit (Promega). .. The sequencing library was prepared using the NEBNext Ultra RNA Library Prep Kit reagent.

    Flow Cytometry:

    Article Title: Abnormal neutrophil signature in the blood and pancreas of presymptomatic and symptomatic type 1 diabetes
    Article Snippet: The cDNA library was prepared using a NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs) following the manufacturer’s instructions. .. The cDNA library was prepared using a NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs) following the manufacturer’s instructions.

    Purification:

    Article Title: Small non-coding RNA landscape of extracellular vesicles from human stem cells
    Article Snippet: Good performance of Exiqons EV isolation kit has been reported . .. NGS libraries were prepared using the NEBNext® Small RNA Library preparation kit (New England Biolabs), consisting of adapter ligation, cDNA conversion, PCR amplification (18 cycles) and purification. .. From a total of 50 µl isolated RNA, 6 μl was converted into microRNA NGS libraries.

    Article Title: Whole body transcriptomes and new insights into the biology of the tick Ixodes ricinus
    Article Snippet: The library preparation kit was NEBNext® Ultra Directional RNA Library Prep Kit, NEB Art. .. The library preparation kit was NEBNext® Ultra Directional RNA Library Prep Kit, NEB Art.

    Article Title: Transforming growth factor beta1 targets estrogen receptor signaling in bronchial epithelial cells
    Article Snippet: 2 μL of 1:2000 diluted External RNA Controls Consortium (ERCC) Spike-In Mix (Ambion™ 4,456,740, ThermoFisher Scientific Inc.) was added to 100 ng of high-quality total RNA followed by mRNA isolation using NEBNext Poly(A) mRNA Magnetic Isolation module (New England Biolabs E7490, Ipswich, MA) and RNA library construction with NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs E7530) according to the manufacturer’s user guide. .. 2 μL of 1:2000 diluted External RNA Controls Consortium (ERCC) Spike-In Mix (Ambion™ 4,456,740, ThermoFisher Scientific Inc.) was added to 100 ng of high-quality total RNA followed by mRNA isolation using NEBNext Poly(A) mRNA Magnetic Isolation module (New England Biolabs E7490, Ipswich, MA) and RNA library construction with NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs E7530) according to the manufacturer’s user guide.

    Article Title: Transcripts of antibacterial peptides in chicken erythrocytes infected with Marek’s disease virus
    Article Snippet: A total of 1 μg purified RNA per sample was used for next-generation sequencing (NGS). .. Sequencing libraries were generated using NEBNext®Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer’s instruction, and index codes were added to attribute sequences to each sample as described previously [ ].

    Polymerase Chain Reaction:

    Article Title: Small non-coding RNA landscape of extracellular vesicles from human stem cells
    Article Snippet: Good performance of Exiqons EV isolation kit has been reported . .. NGS libraries were prepared using the NEBNext® Small RNA Library preparation kit (New England Biolabs), consisting of adapter ligation, cDNA conversion, PCR amplification (18 cycles) and purification. .. From a total of 50 µl isolated RNA, 6 μl was converted into microRNA NGS libraries.

    Article Title: Whole body transcriptomes and new insights into the biology of the tick Ixodes ricinus
    Article Snippet: The library preparation kit was NEBNext® Ultra Directional RNA Library Prep Kit, NEB Art. .. The library preparation kit was NEBNext® Ultra Directional RNA Library Prep Kit, NEB Art.

    Lysis:

    Article Title: Small non-coding RNA landscape of extracellular vesicles from human stem cells
    Article Snippet: Before precipitation, two centrifugation steps were used to remove cellular components and two supernantant removal steps to get rid of the un-precipitated material, followed by EV lysis and RNA isolation using miRCURY™ RNA Isolation Kits - Cell & Plant (Exiqon A/S). .. NGS libraries were prepared using the NEBNext® Small RNA Library preparation kit (New England Biolabs), consisting of adapter ligation, cDNA conversion, PCR amplification (18 cycles) and purification.

    IA:

    Article Title: Mating-Induced Transcriptome Changes in the Reproductive Tract of Female Aedes aegypti
    Article Snippet: The concentration of RNA in each sample was measured using a Qubit spectrophotometer (Invitrogen, Grand Island, NY), and the quality of RNA was measured on a Fragment Analyzer (Advanced Analytical Technologies, Inc., Ames, IA). .. From total RNA, mRNA was extracted using the poly-A mRNA Magnetic Isolation Module (New England Biolabs, Inc., Ipswich, MA). cDNA libraries for each replicate were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs, Inc., Ipswich, MA).

    Plasmid Preparation:

    Article Title: Potent and Targeted Activation of Latent HIV-1 Using the CRISPR/dCas9 Activator Complex
    Article Snippet: CEM LChIT 3.2 bimodal cells were co-transfected with the plasmid pHAGE EF1α dCas9-VP64 together with sg362F or control sgCTRL and a GFP-expressing plasmid as described above. .. The sequencing library was prepared using the NEBNext Ultra RNA Library Prep Kit reagent.

    Software:

    Article Title: In Utero Heat Stress Alters the Offspring Epigenome
    Article Snippet: RNA-Seq library was constructed using NEBNext® Ultra™ RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA) following manufacturer’s recommendations. .. RNA-Seq library was constructed using NEBNext® Ultra™ RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA) following manufacturer’s recommendations.

    Electrophoresis:

    Article Title: Functional Insights into the Roles of Hormones in the Dendrobium officinale-Tulasnella sp. Germinated Seed Symbiotic Association
    Article Snippet: RNA degradation and contamination was monitored by electrophoresis on 1% agarose gels. .. Sequencing libraries were generated using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, Ipswitch, MA, USA) following the manufacturer’s recommendations, and index codes were added to attribute the sequencing reads to each sample.

    RNA Extraction:

    Article Title: Generation and classification of transcriptomes in two Croomia species and molecular evolution of CYC/TB1 genes in Stemonaceae
    Article Snippet: Fresh flowers of C. heterosepala (ChFlower) and C. japonica (CjFlower) at the anthesis stage, as well as juvenile leaf samples of C. japonica (CjLeaf), were harvested from these living plants and immediately frozen in liquid nitrogen for storage at −80 °C until total RNA extraction. .. Sequencing libraries were generated from 3 μg total RNA using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's protocol, and index codes were added to attribute sequences to each sample.

    Article Title: In Utero Heat Stress Alters the Offspring Epigenome
    Article Snippet: A portion of the mammary biopsies from all IUHT-H and IUCL-H were stored at −80 °C in RNALater until RNA extraction for RNA sequencing. .. RNA-Seq library was constructed using NEBNext® Ultra™ RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA) following manufacturer’s recommendations.

    Article Title: Transcriptome-Wide Analysis of Hepatitis B Virus-Mediated Changes to Normal Hepatocyte Gene Expression
    Article Snippet: Paragraph title: RNA extraction, cDNA synthesis, and sequencing ... Total RNA was divided for use in downstream miRNA analyses (see below) or for quality control, polyA selection, cDNA library preparation, and the first round of sequencing by Genewiz, Inc (South Plainfield, NJ). cDNA library preparation was done using the NEBNext Ultra RNA Library Preparation Kit (New England Biolabs, Ipswich, MA), and the first round of sequencing was done using the Illumina HiSeq2500 platform to generate 1 x 50bp reads.

    Article Title: Mating-Induced Transcriptome Changes in the Reproductive Tract of Female Aedes aegypti
    Article Snippet: Paragraph title: RNA extraction, library preparation and read processing ... From total RNA, mRNA was extracted using the poly-A mRNA Magnetic Isolation Module (New England Biolabs, Inc., Ipswich, MA). cDNA libraries for each replicate were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs, Inc., Ipswich, MA).

    Selection:

    Article Title: Whole body transcriptomes and new insights into the biology of the tick Ixodes ricinus
    Article Snippet: The library preparation kit was NEBNext® Ultra Directional RNA Library Prep Kit, NEB Art. .. The library preparation kit was NEBNext® Ultra Directional RNA Library Prep Kit, NEB Art.

    Article Title: Transcriptome-Wide Analysis of Hepatitis B Virus-Mediated Changes to Normal Hepatocyte Gene Expression
    Article Snippet: The PRHs in the secondary dataset were infected with either AdGFP or AdGFP-HBV 24hr after plating. .. Total RNA was divided for use in downstream miRNA analyses (see below) or for quality control, polyA selection, cDNA library preparation, and the first round of sequencing by Genewiz, Inc (South Plainfield, NJ). cDNA library preparation was done using the NEBNext Ultra RNA Library Preparation Kit (New England Biolabs, Ipswich, MA), and the first round of sequencing was done using the Illumina HiSeq2500 platform to generate 1 x 50bp reads. .. The cDNA library was then used for a second round of sequencing by the DUCOM CGS using the Illumina NextSeq 500 platform to generate an additional set of 1x75bp reads.

    Ethanol Precipitation:

    Article Title: Mating-Induced Transcriptome Changes in the Reproductive Tract of Female Aedes aegypti
    Article Snippet: RNA was then prepared by chloroform/isopropanol extraction and ethanol precipitation following the manufacturer’s protocol (Life technologies, Grand Island, NY). .. From total RNA, mRNA was extracted using the poly-A mRNA Magnetic Isolation Module (New England Biolabs, Inc., Ipswich, MA). cDNA libraries for each replicate were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs, Inc., Ipswich, MA).

    Next-Generation Sequencing:

    Article Title: Small non-coding RNA landscape of extracellular vesicles from human stem cells
    Article Snippet: Good performance of Exiqons EV isolation kit has been reported . .. NGS libraries were prepared using the NEBNext® Small RNA Library preparation kit (New England Biolabs), consisting of adapter ligation, cDNA conversion, PCR amplification (18 cycles) and purification. .. From a total of 50 µl isolated RNA, 6 μl was converted into microRNA NGS libraries.

    Article Title: Transcripts of antibacterial peptides in chicken erythrocytes infected with Marek’s disease virus
    Article Snippet: A total of 1 μg purified RNA per sample was used for next-generation sequencing (NGS). .. Sequencing libraries were generated using NEBNext®Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer’s instruction, and index codes were added to attribute sequences to each sample as described previously [ ].

    Spectrophotometry:

    Article Title: Generation and classification of transcriptomes in two Croomia species and molecular evolution of CYC/TB1 genes in Stemonaceae
    Article Snippet: RNA quality and quantity were assessed using gel electrophoresis and a NanoDrop spectrophotometer 2000 (Thermo Scientific, Wilmington, DE, USA). .. Sequencing libraries were generated from 3 μg total RNA using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's protocol, and index codes were added to attribute sequences to each sample.

    Article Title: Functional Insights into the Roles of Hormones in the Dendrobium officinale-Tulasnella sp. Germinated Seed Symbiotic Association
    Article Snippet: RNA purity was checked using the NanoPhotometer® spectrophotometer (Implen, Westlake Vilagge, CA, USA). .. Sequencing libraries were generated using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, Ipswitch, MA, USA) following the manufacturer’s recommendations, and index codes were added to attribute the sequencing reads to each sample.

    Article Title: Mating-Induced Transcriptome Changes in the Reproductive Tract of Female Aedes aegypti
    Article Snippet: The concentration of RNA in each sample was measured using a Qubit spectrophotometer (Invitrogen, Grand Island, NY), and the quality of RNA was measured on a Fragment Analyzer (Advanced Analytical Technologies, Inc., Ames, IA). .. From total RNA, mRNA was extracted using the poly-A mRNA Magnetic Isolation Module (New England Biolabs, Inc., Ipswich, MA). cDNA libraries for each replicate were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs, Inc., Ipswich, MA).

    DNA Methylation Assay:

    Article Title: In Utero Heat Stress Alters the Offspring Epigenome
    Article Snippet: RNA-Seq library was constructed using NEBNext® Ultra™ RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA) following manufacturer’s recommendations. .. RNA-Seq library was constructed using NEBNext® Ultra™ RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA) following manufacturer’s recommendations.

    Concentration Assay:

    Article Title: Transforming growth factor beta1 targets estrogen receptor signaling in bronchial epithelial cells
    Article Snippet: RNA concentration was determined on Qubit® 2.0 Fluorometer (ThermoFisher/Invitrogen, Grand Island, NY), RNA quality was assessed using the Agilent 2100 Bioanalyzer. .. 2 μL of 1:2000 diluted External RNA Controls Consortium (ERCC) Spike-In Mix (Ambion™ 4,456,740, ThermoFisher Scientific Inc.) was added to 100 ng of high-quality total RNA followed by mRNA isolation using NEBNext Poly(A) mRNA Magnetic Isolation module (New England Biolabs E7490, Ipswich, MA) and RNA library construction with NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs E7530) according to the manufacturer’s user guide.

    Article Title: In Utero Heat Stress Alters the Offspring Epigenome
    Article Snippet: Briefly, RNA was extracted from mammary tissue using the RNeasy Plus Universal Mini Kit (Qiagen, Valencia, CA) and RNA concentration was determined using a NanoDrop instrument (ND-2000, ThermoFisher Scientific, Waltham, MA). .. RNA-Seq library was constructed using NEBNext® Ultra™ RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA) following manufacturer’s recommendations.

    Article Title: Studying on the strictly self-compatibility mechanism of ‘Liuyefeitao’ peach (Prunus persica L.)
    Article Snippet: The RNA concentration and purity were checked by OD A260/A280 ( > 1.8) and A260/A230 ( > 1.6), and the yield and quality were accessed using Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and RNA 6000 Nano LabChip Kit (Agilent, CA, USA), RIN > 7.0. .. Transcriptome libraries were constructed using NEBNext® Ultra™ RNA Library Prep Kit for Illumina (New England Biolabs) according to the manufacturer’s instructions.

    Article Title: Comparative transcriptomics identifies genes differentially expressed in the intestine of a new fast-growing strain of common carp with higher unsaturated fatty acid content in muscle
    Article Snippet: To ensure that foregut, midgut and hindgut segments are represented in the transcriptome, RNA was extracted (Trizol, Invitrogen, US) separately from the three segments, and equal amounts (of the three segments belonging to the same specimen) were then pooled together. .. RNA integrity and concentration were assessed using an Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). cDNA libraries were generated using NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, USA) following the manufacturer’s recommendations. .. One μg of RNA per sample was used as input material.

    Article Title: Functional Insights into the Roles of Hormones in the Dendrobium officinale-Tulasnella sp. Germinated Seed Symbiotic Association
    Article Snippet: RNA concentration was measured with the Qubit® RNA Assay Kit in the Qubit® 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA). .. Sequencing libraries were generated using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, Ipswitch, MA, USA) following the manufacturer’s recommendations, and index codes were added to attribute the sequencing reads to each sample.

    Article Title: Mating-Induced Transcriptome Changes in the Reproductive Tract of Female Aedes aegypti
    Article Snippet: The concentration of RNA in each sample was measured using a Qubit spectrophotometer (Invitrogen, Grand Island, NY), and the quality of RNA was measured on a Fragment Analyzer (Advanced Analytical Technologies, Inc., Ames, IA). .. From total RNA, mRNA was extracted using the poly-A mRNA Magnetic Isolation Module (New England Biolabs, Inc., Ipswich, MA). cDNA libraries for each replicate were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs, Inc., Ipswich, MA).

    FACS:

    Article Title: Potent and Targeted Activation of Latent HIV-1 Using the CRISPR/dCas9 Activator Complex
    Article Snippet: To control for transfection efficiency, cells were sorted by fluorescence activated cell sorting based on GFP expression using the BD FACSAria II 72 hours posttransfection. .. The sequencing library was prepared using the NEBNext Ultra RNA Library Prep Kit reagent.

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    New England Biolabs ultra directional rna library prep kit
    Hierarchical clustering of expression levels, based on the rank of the count of exon per million mapped reads (CPM). Dendrogram represents Spearman correlation coefficients between pairs of samples. NEB: <t>NEBNext®</t> Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of <t>RNA;</t> Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuGEN samples; Green and grey: TruSeq samples.. Color scale: Spearman correlation coefficients
    Ultra Directional Rna Library Prep Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 367 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Hierarchical clustering of expression levels, based on the rank of the count of exon per million mapped reads (CPM). Dendrogram represents Spearman correlation coefficients between pairs of samples. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuGEN samples; Green and grey: TruSeq samples.. Color scale: Spearman correlation coefficients

    Journal: BMC Genomics

    Article Title: A comparative analysis of library prep approaches for sequencing low input translatome samples

    doi: 10.1186/s12864-018-5066-2

    Figure Lengend Snippet: Hierarchical clustering of expression levels, based on the rank of the count of exon per million mapped reads (CPM). Dendrogram represents Spearman correlation coefficients between pairs of samples. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuGEN samples; Green and grey: TruSeq samples.. Color scale: Spearman correlation coefficients

    Article Snippet: NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina with 4 ng of RNA

    Techniques: Expressing

    Hierarchical clustering based on the rank of IP/input value. Dendrogram represents Spearman correlation coefficients between pairs of samples. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuGEN samples; Green and grey: TruSeq samples

    Journal: BMC Genomics

    Article Title: A comparative analysis of library prep approaches for sequencing low input translatome samples

    doi: 10.1186/s12864-018-5066-2

    Figure Lengend Snippet: Hierarchical clustering based on the rank of IP/input value. Dendrogram represents Spearman correlation coefficients between pairs of samples. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuGEN samples; Green and grey: TruSeq samples

    Article Snippet: NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina with 4 ng of RNA

    Techniques:

    Descriptive characteristic of enrichment or depletion profiles as generated by the different library preparation kits. Genes which have at least 20 raw reads in the input samples and a ratio of IP/Input ≥2 or Input/IP ≥2 were used to generate the plots. a Total number of transcripts enriched or depleted. b Percentage of enriched or depleted transcripts grouped into different bins. X-axis: log2(IP/input), Y-axis: percentage of genes in each bin over whole population. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuGEN samples; Green and grey: TruSeq samples

    Journal: BMC Genomics

    Article Title: A comparative analysis of library prep approaches for sequencing low input translatome samples

    doi: 10.1186/s12864-018-5066-2

    Figure Lengend Snippet: Descriptive characteristic of enrichment or depletion profiles as generated by the different library preparation kits. Genes which have at least 20 raw reads in the input samples and a ratio of IP/Input ≥2 or Input/IP ≥2 were used to generate the plots. a Total number of transcripts enriched or depleted. b Percentage of enriched or depleted transcripts grouped into different bins. X-axis: log2(IP/input), Y-axis: percentage of genes in each bin over whole population. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuGEN samples; Green and grey: TruSeq samples

    Article Snippet: NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina with 4 ng of RNA

    Techniques: Generated

    Enrichment profiles and top 50 enriched transcripts. a Enrichment factor of transcripts are sorted in decreasing order based on log2 (IP/input). X-axis:transcripts, Y-axis:log2 value of enrichment (IP/Input). b Boxplot of top 50 enriched transcripts. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuGEN samples; Green and grey: TruSeq samples

    Journal: BMC Genomics

    Article Title: A comparative analysis of library prep approaches for sequencing low input translatome samples

    doi: 10.1186/s12864-018-5066-2

    Figure Lengend Snippet: Enrichment profiles and top 50 enriched transcripts. a Enrichment factor of transcripts are sorted in decreasing order based on log2 (IP/input). X-axis:transcripts, Y-axis:log2 value of enrichment (IP/Input). b Boxplot of top 50 enriched transcripts. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuGEN samples; Green and grey: TruSeq samples

    Article Snippet: NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina with 4 ng of RNA

    Techniques:

    Descriptive characteristics of raw and mapped reads. a Total number of raw reads and number of reads mapped to the mouse genome (mm10, GRCm38.84). b Percentage of reads mapped to exonic, intronic and intergenic regions. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA

    Journal: BMC Genomics

    Article Title: A comparative analysis of library prep approaches for sequencing low input translatome samples

    doi: 10.1186/s12864-018-5066-2

    Figure Lengend Snippet: Descriptive characteristics of raw and mapped reads. a Total number of raw reads and number of reads mapped to the mouse genome (mm10, GRCm38.84). b Percentage of reads mapped to exonic, intronic and intergenic regions. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA

    Article Snippet: NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina with 4 ng of RNA

    Techniques:

    Distribution of normalized mean expression of the first (last) 100 bases of transcripts (in 5′- > 3′-orientation). X axis represents the 5′-3′ normalized position; Y axis represents normalized coverage. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuG samples; Green and grey: TruSeq samples. Solid: Input samples. Dotted: Ribo-IP samples

    Journal: BMC Genomics

    Article Title: A comparative analysis of library prep approaches for sequencing low input translatome samples

    doi: 10.1186/s12864-018-5066-2

    Figure Lengend Snippet: Distribution of normalized mean expression of the first (last) 100 bases of transcripts (in 5′- > 3′-orientation). X axis represents the 5′-3′ normalized position; Y axis represents normalized coverage. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuG samples; Green and grey: TruSeq samples. Solid: Input samples. Dotted: Ribo-IP samples

    Article Snippet: NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina with 4 ng of RNA

    Techniques: Expressing

    Venn diagrams of identified features in the different libraries. The features with CPM ≥ 1 in at least one out of 3 replicates were used to generate these plots. a and c represent input samples and b and d represent IP samples. Most transcripts were detected by all kits tested. However, a higher rate of agreement is seen between the NEB, TruSeq and SMART-Seq prepared samples. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA

    Journal: BMC Genomics

    Article Title: A comparative analysis of library prep approaches for sequencing low input translatome samples

    doi: 10.1186/s12864-018-5066-2

    Figure Lengend Snippet: Venn diagrams of identified features in the different libraries. The features with CPM ≥ 1 in at least one out of 3 replicates were used to generate these plots. a and c represent input samples and b and d represent IP samples. Most transcripts were detected by all kits tested. However, a higher rate of agreement is seen between the NEB, TruSeq and SMART-Seq prepared samples. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA

    Article Snippet: NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina with 4 ng of RNA

    Techniques: