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The expression of GAPDH in the four adipose tissues detected by RT-qPCR and <t>RNA-seq.</t> Note: <t>ATFB:</t> adipose tissues of fetal bovines; ATAB: adipose tissues of adult bulls; ATAS: adipose tissues of adult steers; ATAH: adipose tissues of adult heifers. *: P
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1) Product Images from "Characterization of Transcriptional Complexity during Adipose Tissue Development in Bovines of Different Ages and Sexes"

Article Title: Characterization of Transcriptional Complexity during Adipose Tissue Development in Bovines of Different Ages and Sexes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0101261

The expression of GAPDH in the four adipose tissues detected by RT-qPCR and RNA-seq. Note: ATFB: adipose tissues of fetal bovines; ATAB: adipose tissues of adult bulls; ATAS: adipose tissues of adult steers; ATAH: adipose tissues of adult heifers. *: P
Figure Legend Snippet: The expression of GAPDH in the four adipose tissues detected by RT-qPCR and RNA-seq. Note: ATFB: adipose tissues of fetal bovines; ATAB: adipose tissues of adult bulls; ATAS: adipose tissues of adult steers; ATAH: adipose tissues of adult heifers. *: P

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

2) Product Images from "A Versatile and Robust Serine Protease Inhibitor Scaffold from Actinia tenebrosa"

Article Title: A Versatile and Robust Serine Protease Inhibitor Scaffold from Actinia tenebrosa

Journal: Marine Drugs

doi: 10.3390/md17120701

Tissue distribution of ATPI-I in A. tenebrosa . ( A ) Anatomy of a generalized sea anemone (adapted from Reference [ 33 ]), showing the locations of tentacles, acrorhagus, and mesenterial filaments. ( B ) Tissue distribution of messenger RNA (mRNA) of ATPI-I in A. tenebrosa . ATPI-I mRNA is expressed highly in the mesenteric filaments (green) and also present in acrorhagi (red) and to a lesser extent in tentacles (green). FPKM: fragments per kilobase of transcript per million mapped reads. ( C ) MALDI-MS imaging color map showing the distribution of ATPI-I across a section of A. tenebrosa , which was ( D ) subsequently stained with hematoxylin and eosin. Color in ( C ) corresponds to the signal intensity, and scale bars represent 2 mm.
Figure Legend Snippet: Tissue distribution of ATPI-I in A. tenebrosa . ( A ) Anatomy of a generalized sea anemone (adapted from Reference [ 33 ]), showing the locations of tentacles, acrorhagus, and mesenterial filaments. ( B ) Tissue distribution of messenger RNA (mRNA) of ATPI-I in A. tenebrosa . ATPI-I mRNA is expressed highly in the mesenteric filaments (green) and also present in acrorhagi (red) and to a lesser extent in tentacles (green). FPKM: fragments per kilobase of transcript per million mapped reads. ( C ) MALDI-MS imaging color map showing the distribution of ATPI-I across a section of A. tenebrosa , which was ( D ) subsequently stained with hematoxylin and eosin. Color in ( C ) corresponds to the signal intensity, and scale bars represent 2 mm.

Techniques Used: Mass Spectrometry, Imaging, Staining

3) Product Images from "Human PRPF40B regulates hundreds of alternative splicing targets and represses a hypoxia expression signature"

Article Title: Human PRPF40B regulates hundreds of alternative splicing targets and represses a hypoxia expression signature

Journal: RNA

doi: 10.1261/rna.069534.118

mRNA expression of PRPF40B and its paralog PRPF40A in various human cancers by RNA-seq (TCGA). ( A ) In AML, PRPF40A is highly expressed at higher levels than solid tumors, while PRPF40B expression is lower. ( B ) Inverse correlation between PRPF40B and HIF1A mRNA levels in AML ( t -test). ( C ) Positive correlation between PRPF40A and HIF1A mRNA levels in AML ( t -test).
Figure Legend Snippet: mRNA expression of PRPF40B and its paralog PRPF40A in various human cancers by RNA-seq (TCGA). ( A ) In AML, PRPF40A is highly expressed at higher levels than solid tumors, while PRPF40B expression is lower. ( B ) Inverse correlation between PRPF40B and HIF1A mRNA levels in AML ( t -test). ( C ) Positive correlation between PRPF40A and HIF1A mRNA levels in AML ( t -test).

Techniques Used: Expressing, RNA Sequencing Assay

4) Product Images from "Deep mRNA sequencing reveals stage-specific transcriptome alterations during microsclerotia development in the smoke tree vascular wilt pathogen, Verticillium dahliae"

Article Title: Deep mRNA sequencing reveals stage-specific transcriptome alterations during microsclerotia development in the smoke tree vascular wilt pathogen, Verticillium dahliae

Journal: BMC Genomics

doi: 10.1186/1471-2164-15-324

Correlation in expression patterns among the six RNA-sequenced libraries. A . Heatmap of the Pearson correlation of RNA-Seq samples according to gene expression level. Clustering was analyzed based on the expression data of 10,158 genes. B . Principal component analysis (PCA) of the transcriptomes during microsclerotia formation and germinating conidia. For the principal components 1 and 2, eigenvalues are 86.2% and 6.7%, respectively. The analysis was performed by the MultiExperiment Viewer using the expression data from 10,158 genes.
Figure Legend Snippet: Correlation in expression patterns among the six RNA-sequenced libraries. A . Heatmap of the Pearson correlation of RNA-Seq samples according to gene expression level. Clustering was analyzed based on the expression data of 10,158 genes. B . Principal component analysis (PCA) of the transcriptomes during microsclerotia formation and germinating conidia. For the principal components 1 and 2, eigenvalues are 86.2% and 6.7%, respectively. The analysis was performed by the MultiExperiment Viewer using the expression data from 10,158 genes.

Techniques Used: Expressing, RNA Sequencing Assay

Microsclerotia developmental process of the smoke tree vascular wilt fungus, V. dahliae . and the transcriptome analysis pipeline. A . Micrographs showing microsclerotial development processes from conidia to microsclerotia in Verticillium dahliae . The panels represent different developmental stages of smoke tree wilt fungus cultured in CM broth and BM, respectively. Scale bar = 10 μm. MS1-MS4 represent four typical stages during the entire process of microsclerotia formation at 60 h, 72 h, 96 h and 14 d represent four stages of microsclerotia formation used for RNA-Seq. B . Flowcharts of the RNA-Seq method employed in this study. Tophat was used to align RNA-Seq reads to the genome of V. dahliae VdLs.17 (Broad Institute) and Cufflinks package was used to assemble and find differentially expressed genes and transcripts.
Figure Legend Snippet: Microsclerotia developmental process of the smoke tree vascular wilt fungus, V. dahliae . and the transcriptome analysis pipeline. A . Micrographs showing microsclerotial development processes from conidia to microsclerotia in Verticillium dahliae . The panels represent different developmental stages of smoke tree wilt fungus cultured in CM broth and BM, respectively. Scale bar = 10 μm. MS1-MS4 represent four typical stages during the entire process of microsclerotia formation at 60 h, 72 h, 96 h and 14 d represent four stages of microsclerotia formation used for RNA-Seq. B . Flowcharts of the RNA-Seq method employed in this study. Tophat was used to align RNA-Seq reads to the genome of V. dahliae VdLs.17 (Broad Institute) and Cufflinks package was used to assemble and find differentially expressed genes and transcripts.

Techniques Used: Cell Culture, RNA Sequencing Assay

5) Product Images from "Chicken interferome: avian interferon-stimulated genes identified by microarray and RNA-seq of primary chick embryo fibroblasts treated with a chicken type I interferon (IFN-α)"

Article Title: Chicken interferome: avian interferon-stimulated genes identified by microarray and RNA-seq of primary chick embryo fibroblasts treated with a chicken type I interferon (IFN-α)

Journal: Veterinary Research

doi: 10.1186/s13567-016-0363-8

Comparison of expression level and rank of significant ISGs identified by RNA-seq and microarrays. Spearman correlation plots for significant ISGs from: (i) Illumina 100b paired-end RNA-seq and (ii) Affymetrix 32K GeneChip Chicken Genome Array, following induction of CEF for 6 h with 1000 units of rChIFN1, by FC ( A ) and by Rank ( B ).
Figure Legend Snippet: Comparison of expression level and rank of significant ISGs identified by RNA-seq and microarrays. Spearman correlation plots for significant ISGs from: (i) Illumina 100b paired-end RNA-seq and (ii) Affymetrix 32K GeneChip Chicken Genome Array, following induction of CEF for 6 h with 1000 units of rChIFN1, by FC ( A ) and by Rank ( B ).

Techniques Used: Expressing, RNA Sequencing Assay

6) Product Images from "The 25–26 nt Small RNAs in Phytophthora parasitica Are Associated with Efficient Silencing of Homologous Endogenous Genes"

Article Title: The 25–26 nt Small RNAs in Phytophthora parasitica Are Associated with Efficient Silencing of Homologous Endogenous Genes

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2017.00773

The basic characteristic of small RNAs in P. parasitica . (A) The annotation of small RNAs (sRNAs). (B) The length distribution of sRNAs at stages of vegetative mycelial growth and plant infection (24 hpi). The rRNA- and mitochondrial RNA-derived sRNAs were filtered out. (C) The first base preference of sRNAs. (D) The 3′ 2-nt overhang enrichment analysis. Only the sRNAs derived from 25 to 26 nt sRNA clusters and detected in all three mycelial samples were used for overhang enrichment analysis.
Figure Legend Snippet: The basic characteristic of small RNAs in P. parasitica . (A) The annotation of small RNAs (sRNAs). (B) The length distribution of sRNAs at stages of vegetative mycelial growth and plant infection (24 hpi). The rRNA- and mitochondrial RNA-derived sRNAs were filtered out. (C) The first base preference of sRNAs. (D) The 3′ 2-nt overhang enrichment analysis. Only the sRNAs derived from 25 to 26 nt sRNA clusters and detected in all three mycelial samples were used for overhang enrichment analysis.

Techniques Used: Infection, Derivative Assay

7) Product Images from "Correlation of LNCR rasiRNAs Expression with Heterochromatin Formation during Development of the Holocentric Insect Spodoptera frugiperda"

Article Title: Correlation of LNCR rasiRNAs Expression with Heterochromatin Formation during Development of the Holocentric Insect Spodoptera frugiperda

Journal: PLoS ONE

doi: 10.1371/journal.pone.0024746

Northern blot analysis of LNCR expression in S. frugiperda adults. Total RNA (15 micrograms) extracted from S. frugiperda adults was denatured, separated on 1.2% agarose gel (lane 2), together with RNA ladder (lane 1) and stained with EtBr. Molecular sizes of RNA ladder (in nucleotides) are indicated on the left of the figure. The RNA was blotted onto nylon membrane and hybridized with a 32 P-labeled LNCR specific DNA probe. The probe (1244 bp) was generated with ncRNA_long primers (see Material and Methods) by PCR, using cDNA of adults as template. Northern blot is shown in lane 3.
Figure Legend Snippet: Northern blot analysis of LNCR expression in S. frugiperda adults. Total RNA (15 micrograms) extracted from S. frugiperda adults was denatured, separated on 1.2% agarose gel (lane 2), together with RNA ladder (lane 1) and stained with EtBr. Molecular sizes of RNA ladder (in nucleotides) are indicated on the left of the figure. The RNA was blotted onto nylon membrane and hybridized with a 32 P-labeled LNCR specific DNA probe. The probe (1244 bp) was generated with ncRNA_long primers (see Material and Methods) by PCR, using cDNA of adults as template. Northern blot is shown in lane 3.

Techniques Used: Northern Blot, Expressing, Agarose Gel Electrophoresis, Staining, Labeling, Generated, Polymerase Chain Reaction

Expression analysis of S. frugiperda long ncRNA (LNCR) during different developmental stages. A) Expression analysis of S. frugiperda long ncRNA (LNCR) during different developmental stages (E2- 2.5 days old fertilised eggs, L1–L6 larval stages, P- 12-day old pupae and A- adults) by semi quantitative RT PCR. PCR was done on total RNA treated with reverse transcriptase (RT+) or without it (RT−). Expressions of ribosomal gene RPL10A and TBP (TATA binding protein) were used as endogenous control genes. PCR was done with primers amplifying the LNCR region from 27–1271 (upper panel) and LNCR region from 858–1271 nt (lower panel). B) Graph presenting the normalized quantity of expressed LNCR relative to the expression of endogenous control genes: ribosomal gene RPL10A (white bars) and transcription factor TBP (black bars). The quantification was done with ImageQuant TL software. C) PCR done on S. frugiperda genomic DNA with the same pair of primers (pair of primers amplifying the LNCR region from 27–1271 nt) used for expression analysis of LNCR by semi quantitative RT PCR. M indicates DNA ladder and molecular sizes (in base pairs) are shown on the left of the figure.
Figure Legend Snippet: Expression analysis of S. frugiperda long ncRNA (LNCR) during different developmental stages. A) Expression analysis of S. frugiperda long ncRNA (LNCR) during different developmental stages (E2- 2.5 days old fertilised eggs, L1–L6 larval stages, P- 12-day old pupae and A- adults) by semi quantitative RT PCR. PCR was done on total RNA treated with reverse transcriptase (RT+) or without it (RT−). Expressions of ribosomal gene RPL10A and TBP (TATA binding protein) were used as endogenous control genes. PCR was done with primers amplifying the LNCR region from 27–1271 (upper panel) and LNCR region from 858–1271 nt (lower panel). B) Graph presenting the normalized quantity of expressed LNCR relative to the expression of endogenous control genes: ribosomal gene RPL10A (white bars) and transcription factor TBP (black bars). The quantification was done with ImageQuant TL software. C) PCR done on S. frugiperda genomic DNA with the same pair of primers (pair of primers amplifying the LNCR region from 27–1271 nt) used for expression analysis of LNCR by semi quantitative RT PCR. M indicates DNA ladder and molecular sizes (in base pairs) are shown on the left of the figure.

Techniques Used: Expressing, Quantitative RT-PCR, Polymerase Chain Reaction, Binding Assay, Software

Distribution of small RNAs in three developmental stages of S. frugiperda and annotation of mapped rasiRNAs. A) Total RNAs (5 µg) from three developmental stages of S. frugiperda were loaded onto a 17% polyacrylamide gel containing 7 M urea and stained with silver after electrophoretic separation. Molecular sizes (in nucleotides) are shown on the left of the figure. Some discrete bands of small RNAs are labeled with asterisks. 1) Decade small RNA ladder, 2) total RNA 2.5 days old fertilized eggs. 3) total RNA L2 larvae, 4) total RNA 12 days old pupae, 5) small synthetic RNA 21 nt, 6) small synthetic RNA 36 nt. B) Pie charts summarizing the annotation of 102 rasiRNAs, mapped on consensus DNA repeated elements (upper panel) and the annotation of consensus DNA repeated elements defined after analysis of 1% of the sequenced S. frugiperda genome (lower panel).
Figure Legend Snippet: Distribution of small RNAs in three developmental stages of S. frugiperda and annotation of mapped rasiRNAs. A) Total RNAs (5 µg) from three developmental stages of S. frugiperda were loaded onto a 17% polyacrylamide gel containing 7 M urea and stained with silver after electrophoretic separation. Molecular sizes (in nucleotides) are shown on the left of the figure. Some discrete bands of small RNAs are labeled with asterisks. 1) Decade small RNA ladder, 2) total RNA 2.5 days old fertilized eggs. 3) total RNA L2 larvae, 4) total RNA 12 days old pupae, 5) small synthetic RNA 21 nt, 6) small synthetic RNA 36 nt. B) Pie charts summarizing the annotation of 102 rasiRNAs, mapped on consensus DNA repeated elements (upper panel) and the annotation of consensus DNA repeated elements defined after analysis of 1% of the sequenced S. frugiperda genome (lower panel).

Techniques Used: Staining, Labeling

8) Product Images from "Distinct Expression Profiles and Novel Targets of MicroRNAs in Human Spermatogonia, Pachytene Spermatocytes, and Round Spermatids between OA Patients and NOA Patients"

Article Title: Distinct Expression Profiles and Novel Targets of MicroRNAs in Human Spermatogonia, Pachytene Spermatocytes, and Round Spermatids between OA Patients and NOA Patients

Journal: Molecular Therapy. Nucleic Acids

doi: 10.1016/j.omtn.2017.09.007

Morphological and Phenotypic Characterization of Freshly Isolated Human Spermatogonia, Pachytene Spermatocytes, and Round Spermatids (A–C) Phase-contrast microscope revealed the morphology of the freshly isolated human pachytene spermatocytes (A), spermatogonia (B), and round spermatids (C) of OA patients. (D–F) DIC microscope showed the morphological characteristics of the freshly isolated human pachytene spermatocytes (D), spermatogonia (E), and round spermatids (F) of OA patients. Scale bars, 20 μm (A–C) and 5 μm (D–F). (G) RT-PCR revealed the transcripts of GPR125 , RET , GFRA1 , THY1 , UCHL1 , MAGEA4 , and PLZF in the fleshly isolated spermatogonia, the expression of SYCP3 and SYCP1 in pachytene spermatocytes, and mRNA of TNP1 , TNP2 , PRM1 , PRM2 , and ACR in round spermatids. RNA without RT (RT-) but with PCR of GAPDH primers was utilized as negative controls, and GAPDH served as loading controls of total RNA.
Figure Legend Snippet: Morphological and Phenotypic Characterization of Freshly Isolated Human Spermatogonia, Pachytene Spermatocytes, and Round Spermatids (A–C) Phase-contrast microscope revealed the morphology of the freshly isolated human pachytene spermatocytes (A), spermatogonia (B), and round spermatids (C) of OA patients. (D–F) DIC microscope showed the morphological characteristics of the freshly isolated human pachytene spermatocytes (D), spermatogonia (E), and round spermatids (F) of OA patients. Scale bars, 20 μm (A–C) and 5 μm (D–F). (G) RT-PCR revealed the transcripts of GPR125 , RET , GFRA1 , THY1 , UCHL1 , MAGEA4 , and PLZF in the fleshly isolated spermatogonia, the expression of SYCP3 and SYCP1 in pachytene spermatocytes, and mRNA of TNP1 , TNP2 , PRM1 , PRM2 , and ACR in round spermatids. RNA without RT (RT-) but with PCR of GAPDH primers was utilized as negative controls, and GAPDH served as loading controls of total RNA.

Techniques Used: Isolation, Microscopy, Reverse Transcription Polymerase Chain Reaction, Expressing, Polymerase Chain Reaction

9) Product Images from "miRNA alteration is an important mechanism in sugarcane response to low-temperature environment"

Article Title: miRNA alteration is an important mechanism in sugarcane response to low-temperature environment

Journal: BMC Genomics

doi: 10.1186/s12864-017-4231-3

qRT-PCR validation of 13 randomly selected miRNAs identified by small RNA sequencing. a ) miRNAs expressed in FN39. b ) miRNAs expressed in ROC22. Sugarcane buds of FN39 and ROC22 treated with cold stress for 48 h were used as qRT-PCR samples. The data of qRT-PCR were normalized to the 18S rRNA expression level and represented as means of three replicates (n = 3) ± standard error
Figure Legend Snippet: qRT-PCR validation of 13 randomly selected miRNAs identified by small RNA sequencing. a ) miRNAs expressed in FN39. b ) miRNAs expressed in ROC22. Sugarcane buds of FN39 and ROC22 treated with cold stress for 48 h were used as qRT-PCR samples. The data of qRT-PCR were normalized to the 18S rRNA expression level and represented as means of three replicates (n = 3) ± standard error

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

The length distribution of small RNAs in 4 small RNA libraries. F0 and F3 represent the bud samples from sugarcane cultivar FN39 with 0 and 3 h cold (4 °C) treatment, respectively, while R0 and R3 represent the similar samples from cultivar ROC22 with 0 and 3 h cold (4 °C) treatment, respectively
Figure Legend Snippet: The length distribution of small RNAs in 4 small RNA libraries. F0 and F3 represent the bud samples from sugarcane cultivar FN39 with 0 and 3 h cold (4 °C) treatment, respectively, while R0 and R3 represent the similar samples from cultivar ROC22 with 0 and 3 h cold (4 °C) treatment, respectively

Techniques Used:

Scatter plots of differentially expressed miRNAs in 4 small RNA libraries. a ) Known miRNAs; b ) novel miRNAs. Green: significantly downregulated miRNAs; red: significantly upregulated miRNAs; blue: differentially expressed miRNAs (not significant). F0 and F3 represent the bud samples from sugarcane cultivar FN39 with 0 and 3 h cold (4 °C) treatment, respectively, while R0 and R3 represent the similar samples from cultivar ROC22 with 0 and 3 h cold (4 °C) treatment, respectively
Figure Legend Snippet: Scatter plots of differentially expressed miRNAs in 4 small RNA libraries. a ) Known miRNAs; b ) novel miRNAs. Green: significantly downregulated miRNAs; red: significantly upregulated miRNAs; blue: differentially expressed miRNAs (not significant). F0 and F3 represent the bud samples from sugarcane cultivar FN39 with 0 and 3 h cold (4 °C) treatment, respectively, while R0 and R3 represent the similar samples from cultivar ROC22 with 0 and 3 h cold (4 °C) treatment, respectively

Techniques Used:

10) Product Images from "Sendai Virus Infection Induces Expression of Novel RNAs in Human Cells"

Article Title: Sendai Virus Infection Induces Expression of Novel RNAs in Human Cells

Journal: Scientific Reports

doi: 10.1038/s41598-018-35231-8

Comparison of protein coding potential and vertebrate sequence conservation of Sendai virus-induced previously-annotated RNAs and nviRNAs. ( a ) The cumulative distribution frequency of PhyloCSF scores for previously-annotated RNAs (blue) and nviRNAs (red). ( b ) The cumulative distribution frequency of PhyloCSF scores for RNAs mapping to the different genomic feature annotations for previously RefSeq-annotated RNAs and ( c ) nviRNAs. The different genomic feature annotations include: coding exons (orange), intergenic regions (blue), introns (green), and promoters, transcriptional termination sites, and UTRs (all grouped together; red). ( d ) The cumulative distribution frequency of the mean PhastCons score for the previously-annotated RNAs (blue) and nviRNAs (red). The mean PhastCons score for each RNA is plotted against the log 2 fold change in expression after Sendai virus infection for ( e ) previously-annotated RNAs and ( f ) nviRNAs. The 10 most highly-induced previously-annotated RNAs are labeled in blue in ( e ).
Figure Legend Snippet: Comparison of protein coding potential and vertebrate sequence conservation of Sendai virus-induced previously-annotated RNAs and nviRNAs. ( a ) The cumulative distribution frequency of PhyloCSF scores for previously-annotated RNAs (blue) and nviRNAs (red). ( b ) The cumulative distribution frequency of PhyloCSF scores for RNAs mapping to the different genomic feature annotations for previously RefSeq-annotated RNAs and ( c ) nviRNAs. The different genomic feature annotations include: coding exons (orange), intergenic regions (blue), introns (green), and promoters, transcriptional termination sites, and UTRs (all grouped together; red). ( d ) The cumulative distribution frequency of the mean PhastCons score for the previously-annotated RNAs (blue) and nviRNAs (red). The mean PhastCons score for each RNA is plotted against the log 2 fold change in expression after Sendai virus infection for ( e ) previously-annotated RNAs and ( f ) nviRNAs. The 10 most highly-induced previously-annotated RNAs are labeled in blue in ( e ).

Techniques Used: Sequencing, Expressing, Infection, Labeling

nviRNA expression in 2fTGH cells. Total RNA from 2fTGH cells infected with 5 pfu/cell Sendai virus (4 hours) or influenza A virus (IAV;10 hours), or transfected with synthetic dsRNA polyI:C (pI:C; 6 hours), or directly treated with IFNα (6 hours) was analyzed by RT-qPCR. Gene-specific primers were used for ( a ) control genes, ( b ) nviRNAs inducible by viruses and IFNα in Namalwa cells and ( c ) nviRNAs only inducible by viruses in Namalwa cells. Data is representative of ≥2 replicate experiments and is shown normalized to GAPDH expression. Bars indicate average values of technical replicates (n = 3) with error bars representing standard deviation. Statistical analysis was done using a two-tailed Student’s t -test (*p-value
Figure Legend Snippet: nviRNA expression in 2fTGH cells. Total RNA from 2fTGH cells infected with 5 pfu/cell Sendai virus (4 hours) or influenza A virus (IAV;10 hours), or transfected with synthetic dsRNA polyI:C (pI:C; 6 hours), or directly treated with IFNα (6 hours) was analyzed by RT-qPCR. Gene-specific primers were used for ( a ) control genes, ( b ) nviRNAs inducible by viruses and IFNα in Namalwa cells and ( c ) nviRNAs only inducible by viruses in Namalwa cells. Data is representative of ≥2 replicate experiments and is shown normalized to GAPDH expression. Bars indicate average values of technical replicates (n = 3) with error bars representing standard deviation. Statistical analysis was done using a two-tailed Student’s t -test (*p-value

Techniques Used: Expressing, Infection, Transfection, Quantitative RT-PCR, Standard Deviation, Two Tailed Test

Classification of nviRNA expression in Namalwa cells from various stimuli. Total RNA from Namalwa cells infected with 5 pfu/cell for 10 hours of Sendai virus (SeV), influenza A virus, or HSV-1 or directly treated with 1000 U/mL of IFNα for 6 hours was analyzed by RT-qPCR. Heat map indicates expression of each RNA after infection or treatment with IFNα. Average values (n = 3) of fold change are reported normalized to GAPDH expression.
Figure Legend Snippet: Classification of nviRNA expression in Namalwa cells from various stimuli. Total RNA from Namalwa cells infected with 5 pfu/cell for 10 hours of Sendai virus (SeV), influenza A virus, or HSV-1 or directly treated with 1000 U/mL of IFNα for 6 hours was analyzed by RT-qPCR. Heat map indicates expression of each RNA after infection or treatment with IFNα. Average values (n = 3) of fold change are reported normalized to GAPDH expression.

Techniques Used: Expressing, Infection, Quantitative RT-PCR

11) Product Images from "RNA-Seq analyses reveal the order of tRNA processing events and the maturation of C/D box and CRISPR RNAs in the hyperthermophile Methanopyrus kandleri"

Article Title: RNA-Seq analyses reveal the order of tRNA processing events and the maturation of C/D box and CRISPR RNAs in the hyperthermophile Methanopyrus kandleri

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkt317

Detection of crRNA abundance and processing. ( A–E ) Illumina Hiseq2000 reads of T4 PNK-treated (black line) and untreated (gray dashed line) RNA samples were mapped to CRISPR loci 1–5 in the M. kandleri genome. The number of identified processed crRNAs and the detected transcription start site (TSS) are indicated. ( F ) Shown is the consensus repeat sequence and proposed structure. The detected cleavage site is indicated, and mature crRNAs contain an 8 nt 5′ tag. ( G ) A sequence logo of the aligned regions upstream of the first repeat in CRISPR loci 1–5 highlights a conserved transcription start site (TSS) and a boxA promoter sequence.
Figure Legend Snippet: Detection of crRNA abundance and processing. ( A–E ) Illumina Hiseq2000 reads of T4 PNK-treated (black line) and untreated (gray dashed line) RNA samples were mapped to CRISPR loci 1–5 in the M. kandleri genome. The number of identified processed crRNAs and the detected transcription start site (TSS) are indicated. ( F ) Shown is the consensus repeat sequence and proposed structure. The detected cleavage site is indicated, and mature crRNAs contain an 8 nt 5′ tag. ( G ) A sequence logo of the aligned regions upstream of the first repeat in CRISPR loci 1–5 highlights a conserved transcription start site (TSS) and a boxA promoter sequence.

Techniques Used: CRISPR, Sequencing

12) Product Images from "miRNA alteration is an important mechanism in sugarcane response to low-temperature environment"

Article Title: miRNA alteration is an important mechanism in sugarcane response to low-temperature environment

Journal: BMC Genomics

doi: 10.1186/s12864-017-4231-3

qRT-PCR validation of 13 randomly selected miRNAs identified by small RNA sequencing. a ) miRNAs expressed in FN39. b ) miRNAs expressed in ROC22. Sugarcane buds of FN39 and ROC22 treated with cold stress for 48 h were used as qRT-PCR samples. The data of qRT-PCR were normalized to the 18S rRNA expression level and represented as means of three replicates (n = 3) ± standard error
Figure Legend Snippet: qRT-PCR validation of 13 randomly selected miRNAs identified by small RNA sequencing. a ) miRNAs expressed in FN39. b ) miRNAs expressed in ROC22. Sugarcane buds of FN39 and ROC22 treated with cold stress for 48 h were used as qRT-PCR samples. The data of qRT-PCR were normalized to the 18S rRNA expression level and represented as means of three replicates (n = 3) ± standard error

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

Scatter plots of differentially expressed miRNAs in 4 small RNA libraries. a ) Known miRNAs; b ) novel miRNAs. Green: significantly downregulated miRNAs; red: significantly upregulated miRNAs; blue: differentially expressed miRNAs (not significant). F0 and F3 represent the bud samples from sugarcane cultivar FN39 with 0 and 3 h cold (4 °C) treatment, respectively, while R0 and R3 represent the similar samples from cultivar ROC22 with 0 and 3 h cold (4 °C) treatment, respectively
Figure Legend Snippet: Scatter plots of differentially expressed miRNAs in 4 small RNA libraries. a ) Known miRNAs; b ) novel miRNAs. Green: significantly downregulated miRNAs; red: significantly upregulated miRNAs; blue: differentially expressed miRNAs (not significant). F0 and F3 represent the bud samples from sugarcane cultivar FN39 with 0 and 3 h cold (4 °C) treatment, respectively, while R0 and R3 represent the similar samples from cultivar ROC22 with 0 and 3 h cold (4 °C) treatment, respectively

Techniques Used:

13) Product Images from "RNA processing in the minimal organism Nanoarchaeum equitans"

Article Title: RNA processing in the minimal organism Nanoarchaeum equitans

Journal: Genome Biology

doi: 10.1186/gb-2012-13-7-r63

RNA abundance in N. equitans . Illumina HiSeq2000 sequencing reads mapped to the N. equitans reference genome (GenBank: NC_005213 , 490885 bp) highlight the abundance of crRNAs and C/D box sRNAs.
Figure Legend Snippet: RNA abundance in N. equitans . Illumina HiSeq2000 sequencing reads mapped to the N. equitans reference genome (GenBank: NC_005213 , 490885 bp) highlight the abundance of crRNAs and C/D box sRNAs.

Techniques Used: Sequencing

14) Product Images from "Identification and characterization of microRNAs from Chinese pollination constant non-astringent persimmon using high-throughput sequencing"

Article Title: Identification and characterization of microRNAs from Chinese pollination constant non-astringent persimmon using high-throughput sequencing

Journal: BMC Plant Biology

doi: 10.1186/s12870-014-0400-6

Identification of known miRNAs in the two small RNA libraries of ‘Eshi No. 1’. A . The number of known miRNAs in the two libraries constructed using fruits collected at 15 and 20 weeks after flowering (WAF). B . Proportion of known miRNAs with different length in the two libraries. C . Nucleotide preference at each position of the known miRNAs. D . Analysis of first nucleotide bias in the miRNAs of different length. E . The number of miRNA members in the 26 families with more than one member.
Figure Legend Snippet: Identification of known miRNAs in the two small RNA libraries of ‘Eshi No. 1’. A . The number of known miRNAs in the two libraries constructed using fruits collected at 15 and 20 weeks after flowering (WAF). B . Proportion of known miRNAs with different length in the two libraries. C . Nucleotide preference at each position of the known miRNAs. D . Analysis of first nucleotide bias in the miRNAs of different length. E . The number of miRNA members in the 26 families with more than one member.

Techniques Used: Construct

15) Product Images from "Analyzing the Complex Regulatory Landscape of Hfq – an Integrative, Multi-Omics Approach"

Article Title: Analyzing the Complex Regulatory Landscape of Hfq – an Integrative, Multi-Omics Approach

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2017.01784

Validation of candidate loci from the global analysis datasets. (A) Comparative mRNA abundance data for selected loci from the hfq transcriptome. (B) Comparative mRNA abundance data for selected loci from the hfq translatome. In each case, log 2 fold-change values are plotted for the hfq mutant versus WT SBW25. qRT-PCR values are presented alongside the corresponding fold-change observed in (A) the RNA-Seq experiment and (B) the Ribo-Seq experiment. The experiments were repeated at least twice. Data represents mean ± SD. (C) Western blots of selected flag-tagged proteins whose abundance changes in the Hfq proteome. The experiments were repeated at least twice. The representative blots are presented.
Figure Legend Snippet: Validation of candidate loci from the global analysis datasets. (A) Comparative mRNA abundance data for selected loci from the hfq transcriptome. (B) Comparative mRNA abundance data for selected loci from the hfq translatome. In each case, log 2 fold-change values are plotted for the hfq mutant versus WT SBW25. qRT-PCR values are presented alongside the corresponding fold-change observed in (A) the RNA-Seq experiment and (B) the Ribo-Seq experiment. The experiments were repeated at least twice. Data represents mean ± SD. (C) Western blots of selected flag-tagged proteins whose abundance changes in the Hfq proteome. The experiments were repeated at least twice. The representative blots are presented.

Techniques Used: Mutagenesis, Quantitative RT-PCR, RNA Sequencing Assay, Western Blot

16) Product Images from "Conserved miRNAs Are Candidate Post-Transcriptional Regulators of Developmental Arrest in Free-Living and Parasitic Nematodes"

Article Title: Conserved miRNAs Are Candidate Post-Transcriptional Regulators of Developmental Arrest in Free-Living and Parasitic Nematodes

Journal: Genome Biology and Evolution

doi: 10.1093/gbe/evt086

Small RNA-seq expression profiles in Caenorhabditis elegans agree with qRT-PCR data. ( A ) Contingency table of expression fold changes of C. elegans dauer versus mixed stage ob tained by Illumina small RNA deep sequencing compared with qRT-PCR data of dauer versus L2m from Karp et al . (2011) classified according to three categories (upregulated, downregulated, and unaffected). Expression fold changes of both data sets are significantly correlated ( P = 1.1 × 10 −5 , χ 2 test). ( B ) Quantitative comparison of expression fold changes obtained by small RNA-seq and qRT-PCR experiments in C. elegans . Names of all miRNAs with a significant expression change of at least 2-fold in both experiments are displayed. Significance of differential miRNA levels in small RNA-seq data between mixed stage and dauer/iL3 was determined by a two-sided binomial test constrained on the total library sizes followed by correction for multiple testing (FDR
Figure Legend Snippet: Small RNA-seq expression profiles in Caenorhabditis elegans agree with qRT-PCR data. ( A ) Contingency table of expression fold changes of C. elegans dauer versus mixed stage ob tained by Illumina small RNA deep sequencing compared with qRT-PCR data of dauer versus L2m from Karp et al . (2011) classified according to three categories (upregulated, downregulated, and unaffected). Expression fold changes of both data sets are significantly correlated ( P = 1.1 × 10 −5 , χ 2 test). ( B ) Quantitative comparison of expression fold changes obtained by small RNA-seq and qRT-PCR experiments in C. elegans . Names of all miRNAs with a significant expression change of at least 2-fold in both experiments are displayed. Significance of differential miRNA levels in small RNA-seq data between mixed stage and dauer/iL3 was determined by a two-sided binomial test constrained on the total library sizes followed by correction for multiple testing (FDR

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

Experimental setup and computational workflow. Life cycles of Caenorhabditis elegans , Pristionchus pacificus , and Strongyloides ratti . ( A ) Under favorable conditions for reproduction, larvae of C. elegans and P. pacificus develop through four larval stages. Under unfavorable environmental conditions, L2 larvae enter dauer diapause. ( B ) Infective larvae of S. ratti develop either directly or after facultative sexual free-living adult generation. Multiplatform small RNA deep sequencing was performed on mixed and dauer stage samples of C. elegans and P. pacificus . Illumina high-throughput profiling was carried out on mixed and infective stages of S. ratti . ( C ) Schematic overview of in house developed miRNA prediction pipeline. ( D ) miRNA gene complement in all three species, including novel gene candidates (note: several genes occur multiple times in the genome).
Figure Legend Snippet: Experimental setup and computational workflow. Life cycles of Caenorhabditis elegans , Pristionchus pacificus , and Strongyloides ratti . ( A ) Under favorable conditions for reproduction, larvae of C. elegans and P. pacificus develop through four larval stages. Under unfavorable environmental conditions, L2 larvae enter dauer diapause. ( B ) Infective larvae of S. ratti develop either directly or after facultative sexual free-living adult generation. Multiplatform small RNA deep sequencing was performed on mixed and dauer stage samples of C. elegans and P. pacificus . Illumina high-throughput profiling was carried out on mixed and infective stages of S. ratti . ( C ) Schematic overview of in house developed miRNA prediction pipeline. ( D ) miRNA gene complement in all three species, including novel gene candidates (note: several genes occur multiple times in the genome).

Techniques Used: Sequencing, High Throughput Screening Assay

17) Product Images from "Mitochondrial 16S rRNA Is Methylated by tRNA Methyltransferase TRMT61B in All Vertebrates"

Article Title: Mitochondrial 16S rRNA Is Methylated by tRNA Methyltransferase TRMT61B in All Vertebrates

Journal: PLoS Biology

doi: 10.1371/journal.pbio.1002557

Nucleotide distribution in reads corresponding to 16S rRNA position 947 in humans. ( A ) RNA-Seq reads from nine species were mapped to their corresponding mtDNA sequence. Notice that species with an adenine in their mtDNA exhibit the RDDs, while species with a thymine do not (species 8–9). Total read coverage of all samples is shown above each species. ( B ) Coverage and nucleotide distribution of deep sequencing of Sus scrofa samples at the orthologue of human 16S rRNA position 947. The right histogram trio stem from an isolated ribosome (enriched for mature rRNA), and the middle trio stem from total mitochondrial RNA (which contains a mixture of mature and premature rRNAs). The left-most trio of histograms stem from mitochondrial DNA. Notice that the level of m 1 A modification increased in the right trio as compared to the middle one. Exact values are available in S1 Data .
Figure Legend Snippet: Nucleotide distribution in reads corresponding to 16S rRNA position 947 in humans. ( A ) RNA-Seq reads from nine species were mapped to their corresponding mtDNA sequence. Notice that species with an adenine in their mtDNA exhibit the RDDs, while species with a thymine do not (species 8–9). Total read coverage of all samples is shown above each species. ( B ) Coverage and nucleotide distribution of deep sequencing of Sus scrofa samples at the orthologue of human 16S rRNA position 947. The right histogram trio stem from an isolated ribosome (enriched for mature rRNA), and the middle trio stem from total mitochondrial RNA (which contains a mixture of mature and premature rRNAs). The left-most trio of histograms stem from mitochondrial DNA. Notice that the level of m 1 A modification increased in the right trio as compared to the middle one. Exact values are available in S1 Data .

Techniques Used: RNA Sequencing Assay, Sequencing, Isolation, Modification

High structural conservation between S . scrofa mitoribosome and E . coli ribosome at position 947. ( A ) The structure of the porcine mitoribosomal large subunit (PDB accession code 4v1a and 4v19) shown from the subunit interface side. The ribosomal RNA is shown in brown and the ribosomal proteins in green. The ribosomal tRNA A-, P-, and E-binding sites are indicated. ( B ) Sticks-and-ribbon representation of interaction between helices H71 and H64 in S . scrofa (brown) mitoribosome or E . coli (turquoise) ribosome (PDB accession code 4ybb). The hydrogen bond that is likely disrupted by an adenine in position 947 is represented as a dashed line. ( C ) The positively charged m 1 A947 stabilizes the structure by interacting with the negatively charged H64 backbone. Numbers refer to the positions of E . coli ribosomal RNA.
Figure Legend Snippet: High structural conservation between S . scrofa mitoribosome and E . coli ribosome at position 947. ( A ) The structure of the porcine mitoribosomal large subunit (PDB accession code 4v1a and 4v19) shown from the subunit interface side. The ribosomal RNA is shown in brown and the ribosomal proteins in green. The ribosomal tRNA A-, P-, and E-binding sites are indicated. ( B ) Sticks-and-ribbon representation of interaction between helices H71 and H64 in S . scrofa (brown) mitoribosome or E . coli (turquoise) ribosome (PDB accession code 4ybb). The hydrogen bond that is likely disrupted by an adenine in position 947 is represented as a dashed line. ( C ) The positively charged m 1 A947 stabilizes the structure by interacting with the negatively charged H64 backbone. Numbers refer to the positions of E . coli ribosomal RNA.

Techniques Used: Binding Assay

18) Product Images from "RNAseq Analysis of the Parasitic Nematode Strongyloides stercoralis Reveals Divergent Regulation of Canonical Dauer Pathways"

Article Title: RNAseq Analysis of the Parasitic Nematode Strongyloides stercoralis Reveals Divergent Regulation of Canonical Dauer Pathways

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0001854

S. stercoralis RNAseq mean library sizes and number of reads aligning to the genome. A total of 21 libraries were derived from polyadenylated RNA and sequenced from seven developmental stages, each in biological triplicate. Paired-end 100 base-pair (bp) reads were generated from the following developmental stages: free-living females (FL Female), post-free-living first-stage larvae (PFL L1), infectious third-stage larvae (L3i), in vivo activated third-stage larvae (L3+), parasitic females (P Female), predominantly ( > 95%) heterogonically developing post-parasitic first-stage larvae (PP L1), and post-parasitic approximately third-stage larvae heterogonically developing to free-living adults and enriched for females (PP L3). The mean number of reads generated per replicate refers to the mean number of 100 bp reads sequenced (black bars) per biological replicate from each developmental stage. The mean number of mapped reads per replicate refers to the mean number of 100 bp reads aligned to S. stercoralis genomic contigs using TopHat (white bars) per biological replicate from each developmental stage. Error bars represent +1 standard deviation.
Figure Legend Snippet: S. stercoralis RNAseq mean library sizes and number of reads aligning to the genome. A total of 21 libraries were derived from polyadenylated RNA and sequenced from seven developmental stages, each in biological triplicate. Paired-end 100 base-pair (bp) reads were generated from the following developmental stages: free-living females (FL Female), post-free-living first-stage larvae (PFL L1), infectious third-stage larvae (L3i), in vivo activated third-stage larvae (L3+), parasitic females (P Female), predominantly ( > 95%) heterogonically developing post-parasitic first-stage larvae (PP L1), and post-parasitic approximately third-stage larvae heterogonically developing to free-living adults and enriched for females (PP L3). The mean number of reads generated per replicate refers to the mean number of 100 bp reads sequenced (black bars) per biological replicate from each developmental stage. The mean number of mapped reads per replicate refers to the mean number of 100 bp reads aligned to S. stercoralis genomic contigs using TopHat (white bars) per biological replicate from each developmental stage. Error bars represent +1 standard deviation.

Techniques Used: Derivative Assay, Generated, In Vivo, Standard Deviation

19) Product Images from "Global Identification of MicroRNAs and Their Targets in Barley under Salinity Stress"

Article Title: Global Identification of MicroRNAs and Their Targets in Barley under Salinity Stress

Journal: PLoS ONE

doi: 10.1371/journal.pone.0137990

Quantitative real-time PCR analysis of miRNAs and their targets under salinity stress and normal condition. Note: The amount of expression was normalized to the level of 16s RNA. The normalized miRNA levels in control were arbitrarily set to 1.
Figure Legend Snippet: Quantitative real-time PCR analysis of miRNAs and their targets under salinity stress and normal condition. Note: The amount of expression was normalized to the level of 16s RNA. The normalized miRNA levels in control were arbitrarily set to 1.

Techniques Used: Real-time Polymerase Chain Reaction, Expressing

Quantitative real-time PCR analysis of parts miRNA identified in barley. Note: The amount of expression was normalized to the level of 16s RNA.
Figure Legend Snippet: Quantitative real-time PCR analysis of parts miRNA identified in barley. Note: The amount of expression was normalized to the level of 16s RNA.

Techniques Used: Real-time Polymerase Chain Reaction, Expressing

20) Product Images from "Single-cell microRNA-mRNA co-sequencing reveals non-genetic heterogeneity and mechanisms of microRNA regulation"

Article Title: Single-cell microRNA-mRNA co-sequencing reveals non-genetic heterogeneity and mechanisms of microRNA regulation

Journal: Nature Communications

doi: 10.1038/s41467-018-07981-6

Experimental workflow. a Overall strategy for profiling miRNA and mRNA from the same single cells using half-cell genomics. It involves cell lysis, half-cell split, followed by small RNA large RNA library preparation. b For miRNA library preparation, a pre-adenylated (APP) 3′ adaptor was used to ligate to the 3′ end of miRNA molecules, followed by digestion of unreacted 3′ adaptor, ligation with 5′ adaptor, RT and PCR amplification. c For mRNA library preparation, first-strand cDNA synthesis was primed by the 3′ SMART-Seq CDS Primer IIA. Template switching at the 5′ end of transcript was performed using the SMART-Seq v4 oligonucleotides. After PCR amplification, cDNA was fragmented using Illumina’s Tagmentation process
Figure Legend Snippet: Experimental workflow. a Overall strategy for profiling miRNA and mRNA from the same single cells using half-cell genomics. It involves cell lysis, half-cell split, followed by small RNA large RNA library preparation. b For miRNA library preparation, a pre-adenylated (APP) 3′ adaptor was used to ligate to the 3′ end of miRNA molecules, followed by digestion of unreacted 3′ adaptor, ligation with 5′ adaptor, RT and PCR amplification. c For mRNA library preparation, first-strand cDNA synthesis was primed by the 3′ SMART-Seq CDS Primer IIA. Template switching at the 5′ end of transcript was performed using the SMART-Seq v4 oligonucleotides. After PCR amplification, cDNA was fragmented using Illumina’s Tagmentation process

Techniques Used: Lysis, Ligation, Polymerase Chain Reaction, Amplification

21) Product Images from "Transcriptome Profiling of microRNA by Next-Gen Deep Sequencing Reveals Known and Novel miRNA Species in the Lipid Fraction of Human Breast Milk"

Article Title: Transcriptome Profiling of microRNA by Next-Gen Deep Sequencing Reveals Known and Novel miRNA Species in the Lipid Fraction of Human Breast Milk

Journal: PLoS ONE

doi: 10.1371/journal.pone.0050564

Electrophoresis of RNA extracted from breast milk samples. RNA integrity gels were employed in assessing for the integrity of RNA species. smRNA bands are shown, and ribosomal RNA identified with blue arrows. Lanes 1–4: Exosome precipitation with Exoquick system on whole fresh breast milk with a 2 hour precipitation (Lanes 1 2) versus precipitation overnight (Lanes 3 4). Lanes 5 6: Trizol reagent smRNA preparation from lipid breast milk fraction. Lanes 7–10: mirVANA smRNA preparation from lipid fractions of fresh (Lanes 7 8) and previously frozen (Lanes 9 10) breast milk.
Figure Legend Snippet: Electrophoresis of RNA extracted from breast milk samples. RNA integrity gels were employed in assessing for the integrity of RNA species. smRNA bands are shown, and ribosomal RNA identified with blue arrows. Lanes 1–4: Exosome precipitation with Exoquick system on whole fresh breast milk with a 2 hour precipitation (Lanes 1 2) versus precipitation overnight (Lanes 3 4). Lanes 5 6: Trizol reagent smRNA preparation from lipid breast milk fraction. Lanes 7–10: mirVANA smRNA preparation from lipid fractions of fresh (Lanes 7 8) and previously frozen (Lanes 9 10) breast milk.

Techniques Used: Electrophoresis

Chromatograms of breast milk preparations. Shown above are chromatograms from RNA demonstrated in gel electrophoresis from Fig. 2. ( A ) ( B ): Whole breast milk (lanes 1 2), ( C ) ( D ): Lipid fraction (milk fat globules) of fresh breast milk (lanes 7 8), ( E ) ( F ): Lipid fraction frozen breast milk (lanes 9 10). These panels demonstrate enrichment for miRNA (blue boxes) in Trizol-treated, mirVANA isolated samples, which minimized the content of ribosomal RNA.
Figure Legend Snippet: Chromatograms of breast milk preparations. Shown above are chromatograms from RNA demonstrated in gel electrophoresis from Fig. 2. ( A ) ( B ): Whole breast milk (lanes 1 2), ( C ) ( D ): Lipid fraction (milk fat globules) of fresh breast milk (lanes 7 8), ( E ) ( F ): Lipid fraction frozen breast milk (lanes 9 10). These panels demonstrate enrichment for miRNA (blue boxes) in Trizol-treated, mirVANA isolated samples, which minimized the content of ribosomal RNA.

Techniques Used: Nucleic Acid Electrophoresis, Isolation

22) Product Images from "TGF-β induces miR-100 and miR-125b but blocks let-7a through LIN28B controlling PDAC progression"

Article Title: TGF-β induces miR-100 and miR-125b but blocks let-7a through LIN28B controlling PDAC progression

Journal: Nature Communications

doi: 10.1038/s41467-018-03962-x

miR-100 and miR-125b target similar transcripts to regulate common pathways. a , b Venn diagrams showing miR-125b ( a ) or miR-100 ( b ) direct targets represented as overlap of the top 4000 transcripts most enriched onto AGO2 from RIP-seq (RIP) and the top 1300 down-regulated transcripts in RNA-seq (DW), after miRNA overexpression. The bar charts indicate the statistical significance of the overlap between the two groups. P -values were calculated using exact hypergeometric probability test. c , d IPA shows the most enriched canonical pathways for miR-125b ( c ) or miR-100 ( d ). Genes from the overlap between RIP and DW were used as input for the analysis. The threshold of significance is indicated by the intermitted line. Positive Z-score (red) indicates that the canonical pathway is activated and negative Z-score (blue) that is inhibited, based on the expression values in the data set. e , f Sub-networks of genes belonging to significantly enriched IPA canonical pathways using the gene signature coming from overlap between RIP and DW regulated by miR-125b ( e ) or miR-100 ( f ). MiRNA–target interactions inferred from RIP-USE are depicted by different line. Node color represents change in gene expression, from the RNA-seq, mediated by the overexpression of each miRNA
Figure Legend Snippet: miR-100 and miR-125b target similar transcripts to regulate common pathways. a , b Venn diagrams showing miR-125b ( a ) or miR-100 ( b ) direct targets represented as overlap of the top 4000 transcripts most enriched onto AGO2 from RIP-seq (RIP) and the top 1300 down-regulated transcripts in RNA-seq (DW), after miRNA overexpression. The bar charts indicate the statistical significance of the overlap between the two groups. P -values were calculated using exact hypergeometric probability test. c , d IPA shows the most enriched canonical pathways for miR-125b ( c ) or miR-100 ( d ). Genes from the overlap between RIP and DW were used as input for the analysis. The threshold of significance is indicated by the intermitted line. Positive Z-score (red) indicates that the canonical pathway is activated and negative Z-score (blue) that is inhibited, based on the expression values in the data set. e , f Sub-networks of genes belonging to significantly enriched IPA canonical pathways using the gene signature coming from overlap between RIP and DW regulated by miR-125b ( e ) or miR-100 ( f ). MiRNA–target interactions inferred from RIP-USE are depicted by different line. Node color represents change in gene expression, from the RNA-seq, mediated by the overexpression of each miRNA

Techniques Used: RNA Sequencing Assay, Over Expression, Indirect Immunoperoxidase Assay, Expressing

23) Product Images from "m6A facilitates hippocampus-dependent learning and memory through Ythdf1"

Article Title: m6A facilitates hippocampus-dependent learning and memory through Ythdf1

Journal: Nature

doi: 10.1038/s41586-018-0666-1

Ythdf1 binding sites and m 6 A sites in the hippocampus of adult mice, and Ythdf1-mediated effects of m 6 A on hippocampal transcriptome and proteome. a , Peak overlap among three biological replicates of Ythdf1-CLIP-seq. b , Validation of immunoprecipitation efficiency for Ythdf1-CLIP-seq. The position of the gel slice cut during the step of protein-RNA complex size selection was indicated in red (see Methods). c , Consensus motif and its P value generated by HOMER 40 of the three sets of hippocampal m 6 A sites from biological replicates of m 6 A-CLIP-seq. d , e , Distribution of m 6 A-CLIP peaks along the different regions of transcripts ( d ) and genome ( e ). f , Functional annotation of m 6 A-modified transcripts in the adult mouse hippocampus (number of mutations in m 6 A-CLIP-seq > = 5, n = 2,922). g , Peak overlap between high-confidence Ythdf1-CLIP peaks and high-confidence m 6 A-CLIP peaks. The percentage of Ythdf1-CLIP peaks overlapped is indicated. h , IGV screenshots of the piled mutated reads for the each of the biological triplicates of Ythdf1-CLIP-seq (red) and m 6 A-CLIP-seq (blue). Three examples of synaptic plasticity transcripts were presented; the overlapped peak regions are highlighted in orange. i , j , Box-plots of mRNA abundance ( i ) and protein abundance ( j ) log 2 fold changes (Δ) between Ythdf1- KO hippocampus and wild-type control for all expressed genes (black), non-Ythdf1-CLIP transcripts (gray), Ythdf1-CLIP targets (red), transcripts with overlapped Ythdf1-CLIP peaks and m 6 A-CLIP peaks (pink), and m 6 A-modified transcripts (blue). Box-plot elements: center line, median; box limits, upper and lower quartiles, whiskers, 1–99%; P values, two-sided unpaired Kolmogorov-Smirnov test; number of genes and 95% CI of mean are indicated for each box ( i , j ).
Figure Legend Snippet: Ythdf1 binding sites and m 6 A sites in the hippocampus of adult mice, and Ythdf1-mediated effects of m 6 A on hippocampal transcriptome and proteome. a , Peak overlap among three biological replicates of Ythdf1-CLIP-seq. b , Validation of immunoprecipitation efficiency for Ythdf1-CLIP-seq. The position of the gel slice cut during the step of protein-RNA complex size selection was indicated in red (see Methods). c , Consensus motif and its P value generated by HOMER 40 of the three sets of hippocampal m 6 A sites from biological replicates of m 6 A-CLIP-seq. d , e , Distribution of m 6 A-CLIP peaks along the different regions of transcripts ( d ) and genome ( e ). f , Functional annotation of m 6 A-modified transcripts in the adult mouse hippocampus (number of mutations in m 6 A-CLIP-seq > = 5, n = 2,922). g , Peak overlap between high-confidence Ythdf1-CLIP peaks and high-confidence m 6 A-CLIP peaks. The percentage of Ythdf1-CLIP peaks overlapped is indicated. h , IGV screenshots of the piled mutated reads for the each of the biological triplicates of Ythdf1-CLIP-seq (red) and m 6 A-CLIP-seq (blue). Three examples of synaptic plasticity transcripts were presented; the overlapped peak regions are highlighted in orange. i , j , Box-plots of mRNA abundance ( i ) and protein abundance ( j ) log 2 fold changes (Δ) between Ythdf1- KO hippocampus and wild-type control for all expressed genes (black), non-Ythdf1-CLIP transcripts (gray), Ythdf1-CLIP targets (red), transcripts with overlapped Ythdf1-CLIP peaks and m 6 A-CLIP peaks (pink), and m 6 A-modified transcripts (blue). Box-plot elements: center line, median; box limits, upper and lower quartiles, whiskers, 1–99%; P values, two-sided unpaired Kolmogorov-Smirnov test; number of genes and 95% CI of mean are indicated for each box ( i , j ).

Techniques Used: Binding Assay, Mouse Assay, Cross-linking Immunoprecipitation, Immunoprecipitation, Selection, Generated, Functional Assay, Modification

24) Product Images from "Cell Cycle–Dependent Regulation and Function of ARGONAUTE1 in Plants [OPEN]"

Article Title: Cell Cycle–Dependent Regulation and Function of ARGONAUTE1 in Plants [OPEN]

Journal: The Plant Cell

doi: 10.1105/tpc.19.00069

tRFs and the Cell Cycle. (A) tRF fold change (FC) accumulation (in log 2 scale) for the DE tRNA-derived sRNAs (*q-value ≤ 0.05, **q-value ≤ 0.01). (B) High abundant tRNAs with DA fragments in each phase of the cell cycle from the AGO1-IP fractions. (C) Size distribution of tRF abundance in different samples. In black are total RNA samples, in red are all AGO1-IP samples, and in green only the DA tRFs from AGO1-IP samples. RPM, reads per million. (D) Logo representation of the most abundant size category (19-mers) of the differentially accumulated tRFs of AGO1-IP samples. (E) TE distribution of the tRF targets identified using PARE data. The two main categories are represented here: DNA and retrotransposons. (F) Retrotransposable elements targeted by tRFs identified using PARE data, divided into superfamilies. Represented here are the percentages of total number of targeted retrotransposons (left) and the normalized number of retrotransposons (right).
Figure Legend Snippet: tRFs and the Cell Cycle. (A) tRF fold change (FC) accumulation (in log 2 scale) for the DE tRNA-derived sRNAs (*q-value ≤ 0.05, **q-value ≤ 0.01). (B) High abundant tRNAs with DA fragments in each phase of the cell cycle from the AGO1-IP fractions. (C) Size distribution of tRF abundance in different samples. In black are total RNA samples, in red are all AGO1-IP samples, and in green only the DA tRFs from AGO1-IP samples. RPM, reads per million. (D) Logo representation of the most abundant size category (19-mers) of the differentially accumulated tRFs of AGO1-IP samples. (E) TE distribution of the tRF targets identified using PARE data. The two main categories are represented here: DNA and retrotransposons. (F) Retrotransposable elements targeted by tRFs identified using PARE data, divided into superfamilies. Represented here are the percentages of total number of targeted retrotransposons (left) and the normalized number of retrotransposons (right).

Techniques Used: Derivative Assay

sRNAs and the Cell Cycle. (A) sRNA abundance per category. Five biological replicates of total RNA samples (left) and three biological replicates of AGO1-IP samples (right). The x axis represents the different sRNA categories included in this study, and the y axis represents their abundance (reads per million [RPM]). (B) miRNA fold change (FC) accumulation (in log 2 scale) for the differentially accumulated (DA) miRNAs (*q-value ≤ 0.05 and **q-value ≤ 0.01). (C) Highly abundant miRNAs in each cell cycle phase. In black are the DA genes identified in total RNA samples, and in blue are those identified in AGO1-IP samples. (D) miRNA-target pairs in each phase of the cell cycle. Colors are indicative of the abundance, with dark gray representing the most abundant and white the least abundant. (E) Overlap of the miRNA targets with genes DE in the RNA-seq. DE, differential expression.
Figure Legend Snippet: sRNAs and the Cell Cycle. (A) sRNA abundance per category. Five biological replicates of total RNA samples (left) and three biological replicates of AGO1-IP samples (right). The x axis represents the different sRNA categories included in this study, and the y axis represents their abundance (reads per million [RPM]). (B) miRNA fold change (FC) accumulation (in log 2 scale) for the differentially accumulated (DA) miRNAs (*q-value ≤ 0.05 and **q-value ≤ 0.01). (C) Highly abundant miRNAs in each cell cycle phase. In black are the DA genes identified in total RNA samples, and in blue are those identified in AGO1-IP samples. (D) miRNA-target pairs in each phase of the cell cycle. Colors are indicative of the abundance, with dark gray representing the most abundant and white the least abundant. (E) Overlap of the miRNA targets with genes DE in the RNA-seq. DE, differential expression.

Techniques Used: RNA Sequencing Assay, Expressing

25) Product Images from "Identification and Expression Profiling of MicroRNAs in the Brain, Liver and Gonads of Marine Medaka (Oryzias melastigma) and in Response to Hypoxia"

Article Title: Identification and Expression Profiling of MicroRNAs in the Brain, Liver and Gonads of Marine Medaka (Oryzias melastigma) and in Response to Hypoxia

Journal: PLoS ONE

doi: 10.1371/journal.pone.0110698

Schematic diagram of the workflow for O. melastigma miRNA discovery. Brain, liver and gonadal (ovary and testis) tissues of male and female marine medaka were submitted to small RNA libraries preparation and were sequenced using Illumina GAIIX. After the removal of low quality reads and filtering, high quality sequencing reads were blasted against miRBase v.17 to identify the conserved miRNAs of marine medaka.
Figure Legend Snippet: Schematic diagram of the workflow for O. melastigma miRNA discovery. Brain, liver and gonadal (ovary and testis) tissues of male and female marine medaka were submitted to small RNA libraries preparation and were sequenced using Illumina GAIIX. After the removal of low quality reads and filtering, high quality sequencing reads were blasted against miRBase v.17 to identify the conserved miRNAs of marine medaka.

Techniques Used: Sequencing

26) Product Images from "Sendai Virus Infection Induces Expression of Novel RNAs in Human Cells"

Article Title: Sendai Virus Infection Induces Expression of Novel RNAs in Human Cells

Journal: Scientific Reports

doi: 10.1038/s41598-018-35231-8

Comparison of protein coding potential and vertebrate sequence conservation of Sendai virus-induced previously-annotated RNAs and nviRNAs. ( a ) The cumulative distribution frequency of PhyloCSF scores for previously-annotated RNAs (blue) and nviRNAs (red). ( b ) The cumulative distribution frequency of PhyloCSF scores for RNAs mapping to the different genomic feature annotations for previously RefSeq-annotated RNAs and ( c ) nviRNAs. The different genomic feature annotations include: coding exons (orange), intergenic regions (blue), introns (green), and promoters, transcriptional termination sites, and UTRs (all grouped together; red). ( d ) The cumulative distribution frequency of the mean PhastCons score for the previously-annotated RNAs (blue) and nviRNAs (red). The mean PhastCons score for each RNA is plotted against the log 2 fold change in expression after Sendai virus infection for ( e ) previously-annotated RNAs and ( f ) nviRNAs. The 10 most highly-induced previously-annotated RNAs are labeled in blue in ( e ).
Figure Legend Snippet: Comparison of protein coding potential and vertebrate sequence conservation of Sendai virus-induced previously-annotated RNAs and nviRNAs. ( a ) The cumulative distribution frequency of PhyloCSF scores for previously-annotated RNAs (blue) and nviRNAs (red). ( b ) The cumulative distribution frequency of PhyloCSF scores for RNAs mapping to the different genomic feature annotations for previously RefSeq-annotated RNAs and ( c ) nviRNAs. The different genomic feature annotations include: coding exons (orange), intergenic regions (blue), introns (green), and promoters, transcriptional termination sites, and UTRs (all grouped together; red). ( d ) The cumulative distribution frequency of the mean PhastCons score for the previously-annotated RNAs (blue) and nviRNAs (red). The mean PhastCons score for each RNA is plotted against the log 2 fold change in expression after Sendai virus infection for ( e ) previously-annotated RNAs and ( f ) nviRNAs. The 10 most highly-induced previously-annotated RNAs are labeled in blue in ( e ).

Techniques Used: Sequencing, Expressing, Infection, Labeling

nviRNA expression in THP-1 cells. Total RNA from THP-1 cells infected with 5 pfu/cell Sendai virus (4 hours), influenza A virus (IAV; 10 hours) or HSV-1 (10 hours) or directly treated with IFNα (6 hours) was analyzed by RT-qPCR. Gene-specific primers were used for ( a ) control genes, ( b ) nviRNAs inducible by viruses and IFNα in Namalwa cells and ( c ) nviRNAs only inducible by viruses in Namalwa cells. Data is representative of ≥2 replicate experiments and is shown normalized to GAPDH expression. Bars indicate average values of technical replicates (n = 3) with error bars representing standard deviation. Statistical analysis was done using a two-tailed Student’s t -test (*p-value
Figure Legend Snippet: nviRNA expression in THP-1 cells. Total RNA from THP-1 cells infected with 5 pfu/cell Sendai virus (4 hours), influenza A virus (IAV; 10 hours) or HSV-1 (10 hours) or directly treated with IFNα (6 hours) was analyzed by RT-qPCR. Gene-specific primers were used for ( a ) control genes, ( b ) nviRNAs inducible by viruses and IFNα in Namalwa cells and ( c ) nviRNAs only inducible by viruses in Namalwa cells. Data is representative of ≥2 replicate experiments and is shown normalized to GAPDH expression. Bars indicate average values of technical replicates (n = 3) with error bars representing standard deviation. Statistical analysis was done using a two-tailed Student’s t -test (*p-value

Techniques Used: Expressing, Infection, Quantitative RT-PCR, Standard Deviation, Two Tailed Test

Classification of nviRNA expression in Namalwa cells from various stimuli. Total RNA from Namalwa cells infected with 5 pfu/cell for 10 hours of Sendai virus (SeV), influenza A virus, or HSV-1 or directly treated with 1000 U/mL of IFNα for 6 hours was analyzed by RT-qPCR. Heat map indicates expression of each RNA after infection or treatment with IFNα. Average values (n = 3) of fold change are reported normalized to GAPDH expression.
Figure Legend Snippet: Classification of nviRNA expression in Namalwa cells from various stimuli. Total RNA from Namalwa cells infected with 5 pfu/cell for 10 hours of Sendai virus (SeV), influenza A virus, or HSV-1 or directly treated with 1000 U/mL of IFNα for 6 hours was analyzed by RT-qPCR. Heat map indicates expression of each RNA after infection or treatment with IFNα. Average values (n = 3) of fold change are reported normalized to GAPDH expression.

Techniques Used: Expressing, Infection, Quantitative RT-PCR

Validation of differential gene expression induced by Sendai virus infection. Namalwa cells were infected with Sendai virus for 6 hours and total RNA was analyzed by RNA-sequencing or by RT-qPCR in independent samples. For each indicated RNA, RNA-sequencing counts are plotted on the graph on the left and RNA abundance from RT-qPCR on the right. Expression of virus-induced ( a ) previously-annotated genes and ( b ) previously-unannotated RNAs and ( c ) virus-suppressed genes was validated. RNA abundance data is representative of ≥2 replicate experiments and is shown normalized to GAPDH expression. Bars indicate average values of technical replicates (n = 3) with error bars representing standard deviation. Statistical analysis was done using a two-tailed Student’s t -test for RT-qPCR measurements (*p-value
Figure Legend Snippet: Validation of differential gene expression induced by Sendai virus infection. Namalwa cells were infected with Sendai virus for 6 hours and total RNA was analyzed by RNA-sequencing or by RT-qPCR in independent samples. For each indicated RNA, RNA-sequencing counts are plotted on the graph on the left and RNA abundance from RT-qPCR on the right. Expression of virus-induced ( a ) previously-annotated genes and ( b ) previously-unannotated RNAs and ( c ) virus-suppressed genes was validated. RNA abundance data is representative of ≥2 replicate experiments and is shown normalized to GAPDH expression. Bars indicate average values of technical replicates (n = 3) with error bars representing standard deviation. Statistical analysis was done using a two-tailed Student’s t -test for RT-qPCR measurements (*p-value

Techniques Used: Expressing, Infection, RNA Sequencing Assay, Quantitative RT-PCR, Standard Deviation, Two Tailed Test

nviRNA expression in 2fTGH cells. Total RNA from 2fTGH cells infected with 5 pfu/cell Sendai virus (4 hours) or influenza A virus (IAV;10 hours), or transfected with synthetic dsRNA polyI:C (pI:C; 6 hours), or directly treated with IFNα (6 hours) was analyzed by RT-qPCR. Gene-specific primers were used for ( a ) control genes, ( b ) nviRNAs inducible by viruses and IFNα in Namalwa cells and ( c ) nviRNAs only inducible by viruses in Namalwa cells. Data is representative of ≥2 replicate experiments and is shown normalized to GAPDH expression. Bars indicate average values of technical replicates (n = 3) with error bars representing standard deviation. Statistical analysis was done using a two-tailed Student’s t -test (*p-value
Figure Legend Snippet: nviRNA expression in 2fTGH cells. Total RNA from 2fTGH cells infected with 5 pfu/cell Sendai virus (4 hours) or influenza A virus (IAV;10 hours), or transfected with synthetic dsRNA polyI:C (pI:C; 6 hours), or directly treated with IFNα (6 hours) was analyzed by RT-qPCR. Gene-specific primers were used for ( a ) control genes, ( b ) nviRNAs inducible by viruses and IFNα in Namalwa cells and ( c ) nviRNAs only inducible by viruses in Namalwa cells. Data is representative of ≥2 replicate experiments and is shown normalized to GAPDH expression. Bars indicate average values of technical replicates (n = 3) with error bars representing standard deviation. Statistical analysis was done using a two-tailed Student’s t -test (*p-value

Techniques Used: Expressing, Infection, Transfection, Quantitative RT-PCR, Standard Deviation, Two Tailed Test

27) Product Images from "Identification of microRNAs in the Toxigenic Dinoflagellate Alexandrium catenella by High-Throughput Illumina Sequencing and Bioinformatic Analysis"

Article Title: Identification of microRNAs in the Toxigenic Dinoflagellate Alexandrium catenella by High-Throughput Illumina Sequencing and Bioinformatic Analysis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0138709

Length distribution and abundance of sequenced small RNA sequences in A . catenella libraries. Among the small RNAs in the range of 18–25 nt, the most abundant size was 24 nt, followed by 22 nt and 25 nt.
Figure Legend Snippet: Length distribution and abundance of sequenced small RNA sequences in A . catenella libraries. Among the small RNAs in the range of 18–25 nt, the most abundant size was 24 nt, followed by 22 nt and 25 nt.

Techniques Used:

28) Product Images from "Integration of Hi-C and ChIP-seq data reveals distinct types of chromatin linkages"

Article Title: Integration of Hi-C and ChIP-seq data reveals distinct types of chromatin linkages

Journal: Nucleic Acids Research

doi: 10.1093/nar/gks501

Schematic model of GATA-regulated chromatin linkages. Dynamic chromatin interactions form globule structures, which may function to initiate or stabilize three-dimensional gene regulatory structures. Multiple CREs, including enhancers and promoters, are bridged by different groups of transcription factors (TFs) and mediators under different conditions. In the example shown, a pair of interacting loci from cluster 9 is represented. A loop is formed between a promoter and a distal region via interactions of GATA1, BRG1, INI1 and SIRT6 (a histone deacetylase), all bound to a region having H3K4me1 but no marks of an active enhancer or promoter; the nearby gene is repressed. On loss of GATA1 (via reduction of levels of the protein by treatment with siRNAs or on normal physiological changes or owing to displacement of GATA1 by another DNA-binding factor), a different set of enhancer-binding factors are recruited to the distal open chromatin region (which now gains the H3K27Ac mark), the loop changes from a repressive to an activating structure, active histone modifications are placed on the promoter region, RNA Polymerase II is recruited and begins transcription, and the transcribed region gains H3K36me3. We note that GATA1 can also activate transcription, and thus other functions of GATA1-mediated loops can be envisioned.
Figure Legend Snippet: Schematic model of GATA-regulated chromatin linkages. Dynamic chromatin interactions form globule structures, which may function to initiate or stabilize three-dimensional gene regulatory structures. Multiple CREs, including enhancers and promoters, are bridged by different groups of transcription factors (TFs) and mediators under different conditions. In the example shown, a pair of interacting loci from cluster 9 is represented. A loop is formed between a promoter and a distal region via interactions of GATA1, BRG1, INI1 and SIRT6 (a histone deacetylase), all bound to a region having H3K4me1 but no marks of an active enhancer or promoter; the nearby gene is repressed. On loss of GATA1 (via reduction of levels of the protein by treatment with siRNAs or on normal physiological changes or owing to displacement of GATA1 by another DNA-binding factor), a different set of enhancer-binding factors are recruited to the distal open chromatin region (which now gains the H3K27Ac mark), the loop changes from a repressive to an activating structure, active histone modifications are placed on the promoter region, RNA Polymerase II is recruited and begins transcription, and the transcribed region gains H3K36me3. We note that GATA1 can also activate transcription, and thus other functions of GATA1-mediated loops can be envisioned.

Techniques Used: Histone Deacetylase Assay, Binding Assay

Gene expression changes induced by GATA1 and GATA2 knockdown. RNA-seq was performed using control cells and cells treated with siRNAs to either GATA1 or GATA2, and genes whose expression was altered upon knockdown were identified. By intersection of the list of deregulated genes with genes having nearby bound GATA factors, direct target genes were identified (blue pie segments). A second set of direct target genes were identified by intersection of the list of deregulated genes with genes linked to a bound GATA factor via chromatin looping (maroon pie segments). All other genes were classified as indirectly regulated genes (green pie segments). The colors in the pie chart correspond to these different categories of GATA-regulated genes, as labelled in left panel. For each category, the percentage of genes that were upregulated versus downregulated in the knockdown cells is shown by the purple and orange closed pie graphs. (G: GATA factor; Pol: RNA Polymerase II, TF: a transcription factor whose expression is regulated by a GATA factor).
Figure Legend Snippet: Gene expression changes induced by GATA1 and GATA2 knockdown. RNA-seq was performed using control cells and cells treated with siRNAs to either GATA1 or GATA2, and genes whose expression was altered upon knockdown were identified. By intersection of the list of deregulated genes with genes having nearby bound GATA factors, direct target genes were identified (blue pie segments). A second set of direct target genes were identified by intersection of the list of deregulated genes with genes linked to a bound GATA factor via chromatin looping (maroon pie segments). All other genes were classified as indirectly regulated genes (green pie segments). The colors in the pie chart correspond to these different categories of GATA-regulated genes, as labelled in left panel. For each category, the percentage of genes that were upregulated versus downregulated in the knockdown cells is shown by the purple and orange closed pie graphs. (G: GATA factor; Pol: RNA Polymerase II, TF: a transcription factor whose expression is regulated by a GATA factor).

Techniques Used: Expressing, RNA Sequencing Assay

29) Product Images from "Widespread splicing changes in human brain development and aging"

Article Title: Widespread splicing changes in human brain development and aging

Journal: Molecular Systems Biology

doi: 10.1038/msb.2012.67

Splicing changes in development and aging. ( A ) Distribution of the inclusion ratio change index for development (solid line) and aging (dash line) for 1484 age-related segments. The negative index represents the inclusion ratio decrease with age, positive index, the inclusion ratio increase. The colors represent RNA-seq data sets analyzed: yellow—DS1/PFC, gray—DS1/CBC, and red—DS2/PFC. ( B ) Scatter plot of the inclusion ratio change showing different splice types: violet—skipped exons, yellow—retained introns, gray and white—complex and mixed splice types respectively. Four quadrants of the plot correspond to four patterns of inclusion ratio change with age: down–up, up–up, up–down and down–down. Inclusion ratio change is plotted for the 853 segments showing significant splicing changes with age in both data sets and showing consistent direction of age-related splicing change between data sets in development, as well as in aging. ( C ) Schematic representation of segment numbers (represented by the size of the pie diagrams) and splicing type fractions (represented by colors) in the four inclusion ratio change patterns shown in panel B. ( D ) Fractions of NMD segments in the four inclusion ratio change patterns. Numbers on top of the bars show the number of NMD segments and the total number of segments in each pattern. Asterisks indicate significant enrichment of NMD segments in the ‘down–up' pattern (one-sided Fisher's exact test, P =0.0002) ( E ) Distribution of the phastCons scores of segments in the four inclusion ratio change patterns. Asterisks indicate significantly low conservation of segments in the ‘down–up' pattern (one-sided Wilcoxon test, P
Figure Legend Snippet: Splicing changes in development and aging. ( A ) Distribution of the inclusion ratio change index for development (solid line) and aging (dash line) for 1484 age-related segments. The negative index represents the inclusion ratio decrease with age, positive index, the inclusion ratio increase. The colors represent RNA-seq data sets analyzed: yellow—DS1/PFC, gray—DS1/CBC, and red—DS2/PFC. ( B ) Scatter plot of the inclusion ratio change showing different splice types: violet—skipped exons, yellow—retained introns, gray and white—complex and mixed splice types respectively. Four quadrants of the plot correspond to four patterns of inclusion ratio change with age: down–up, up–up, up–down and down–down. Inclusion ratio change is plotted for the 853 segments showing significant splicing changes with age in both data sets and showing consistent direction of age-related splicing change between data sets in development, as well as in aging. ( C ) Schematic representation of segment numbers (represented by the size of the pie diagrams) and splicing type fractions (represented by colors) in the four inclusion ratio change patterns shown in panel B. ( D ) Fractions of NMD segments in the four inclusion ratio change patterns. Numbers on top of the bars show the number of NMD segments and the total number of segments in each pattern. Asterisks indicate significant enrichment of NMD segments in the ‘down–up' pattern (one-sided Fisher's exact test, P =0.0002) ( E ) Distribution of the phastCons scores of segments in the four inclusion ratio change patterns. Asterisks indicate significantly low conservation of segments in the ‘down–up' pattern (one-sided Wilcoxon test, P

Techniques Used: RNA Sequencing Assay

Age-related splicing changes in the human brain. ( A ) Sample age distribution in PFC (orange) and CBC (gray). Each dot represents an individual; the darker shades of color represent older age. In DS1, five individuals of similar age were combined in each sample, resulting in six PFC- and six CBC-pooled samples with the average ages marked on the horizontal axis. ( B ) Fraction of genes (right) and segments (left) showing significant age-related splicing changes in DS1 (DS1 age), DS2 (DS2 age), and in both data sets (DS1 and DS2), as well as genes showing significantly different age-related changes in brain regions in DS1 (DS1 tissue:age). ( C ) Correlation of age-related splicing changes between DS1 and DS2. Shown are the distributions of the Pearson correlation coefficients for non-age-related segments (gray, N =22 360); segments significant only in DS2 (yellow, N =4630); segments significant only in DS1 (orange, N =1648); and segments significant in both data sets (red, N =1484). In all cases, only segments with sufficient coverage in both data sets were used. ( D ) Correlation of inclusion ratio changes with age measured by RT–PCR (horizontal axis) and RNA-seq (vertical axis). ( E ) Correlation of inclusion ratio estimates measured by RT–PCR (horizontal axis) and RNA-seq (vertical axis). ( F ) Correlation of age-related splicing changes measured at the transcript (RNA-seq) and protein (mass spectrometry) levels. Shown are the distributions of the Pearson correlation coefficients for the actual observations (orange) and 10 000 random permutations (gray). The difference between the means of observed and permutation distributions was significant ( q =0 .0004). At the individual segment level, 4 of 24 segments showed marginally significant correlations (Pearson correlation test, BH-corrected FDR
Figure Legend Snippet: Age-related splicing changes in the human brain. ( A ) Sample age distribution in PFC (orange) and CBC (gray). Each dot represents an individual; the darker shades of color represent older age. In DS1, five individuals of similar age were combined in each sample, resulting in six PFC- and six CBC-pooled samples with the average ages marked on the horizontal axis. ( B ) Fraction of genes (right) and segments (left) showing significant age-related splicing changes in DS1 (DS1 age), DS2 (DS2 age), and in both data sets (DS1 and DS2), as well as genes showing significantly different age-related changes in brain regions in DS1 (DS1 tissue:age). ( C ) Correlation of age-related splicing changes between DS1 and DS2. Shown are the distributions of the Pearson correlation coefficients for non-age-related segments (gray, N =22 360); segments significant only in DS2 (yellow, N =4630); segments significant only in DS1 (orange, N =1648); and segments significant in both data sets (red, N =1484). In all cases, only segments with sufficient coverage in both data sets were used. ( D ) Correlation of inclusion ratio changes with age measured by RT–PCR (horizontal axis) and RNA-seq (vertical axis). ( E ) Correlation of inclusion ratio estimates measured by RT–PCR (horizontal axis) and RNA-seq (vertical axis). ( F ) Correlation of age-related splicing changes measured at the transcript (RNA-seq) and protein (mass spectrometry) levels. Shown are the distributions of the Pearson correlation coefficients for the actual observations (orange) and 10 000 random permutations (gray). The difference between the means of observed and permutation distributions was significant ( q =0 .0004). At the individual segment level, 4 of 24 segments showed marginally significant correlations (Pearson correlation test, BH-corrected FDR

Techniques Used: Reverse Transcription Polymerase Chain Reaction, RNA Sequencing Assay, Mass Spectrometry

Patterns and regulation of age-related splicing changes. ( A ) Age-related splicing patterns in the PFC (orange) and CBC (gray). The horizontal axis shows the samples' ages in years. The vertical axis shows the inclusion ratio normalized to mean=0 and s.d.=1 for each segment in each brain region. The points represent the mean inclusion ratios of segments within each pattern in each sample. The lines and the error bars show the cubic spline curves of the mean inclusion ratios and the s.d. of the curves, respectively. The colors represent RNA-seq data sets analyzed: yellow—DS1/PFC, gray—DS1/CBC, and red—DS2/PFC. The numbers above the panels show the ID and the numbers of segments and genes in each pattern. The percentages shown within the panels indicate the percentage of splicing variation found in development and in aging. The vertical dotted line indicates separation between developmental and aging intervals. Numbers of segments separately significant in development or in aging are shown at the bottom of the panels. ( B ) Top: numbers of SFs with significant enrichment of SFBM in a given pattern (Fisher's exact test, P
Figure Legend Snippet: Patterns and regulation of age-related splicing changes. ( A ) Age-related splicing patterns in the PFC (orange) and CBC (gray). The horizontal axis shows the samples' ages in years. The vertical axis shows the inclusion ratio normalized to mean=0 and s.d.=1 for each segment in each brain region. The points represent the mean inclusion ratios of segments within each pattern in each sample. The lines and the error bars show the cubic spline curves of the mean inclusion ratios and the s.d. of the curves, respectively. The colors represent RNA-seq data sets analyzed: yellow—DS1/PFC, gray—DS1/CBC, and red—DS2/PFC. The numbers above the panels show the ID and the numbers of segments and genes in each pattern. The percentages shown within the panels indicate the percentage of splicing variation found in development and in aging. The vertical dotted line indicates separation between developmental and aging intervals. Numbers of segments separately significant in development or in aging are shown at the bottom of the panels. ( B ) Top: numbers of SFs with significant enrichment of SFBM in a given pattern (Fisher's exact test, P

Techniques Used: RNA Sequencing Assay

Age-related splicing changes in PFC and CBC. ( A ) Correlation of age-related splicing changes between the PFC and CBC in DS1. Shown are distributions of the Pearson correlation coefficients for 24 458 segments with sufficient coverage in both data sets but not significant in DS1 (gray), and for 3132 segments showing significant age-related splicing changes in DS1 (red). ( B ) Age-related splicing changes in the protocadherin gamma transcript in the human PFC and CBC. RNA-seq coverage in all 12 DS1 samples is shown. ( C) Inclusion ratio profiles of the protocadherin gamma C 3 -C 5 exons. The colors represent the RNA-seq data sets analyzed: yellow—DS1/PFC, gray—DS1/CBC, and red—DS2/PFC. The points represent the inclusion ratios and the lines represent the cubic spline curves fitted with three degrees of freedom.
Figure Legend Snippet: Age-related splicing changes in PFC and CBC. ( A ) Correlation of age-related splicing changes between the PFC and CBC in DS1. Shown are distributions of the Pearson correlation coefficients for 24 458 segments with sufficient coverage in both data sets but not significant in DS1 (gray), and for 3132 segments showing significant age-related splicing changes in DS1 (red). ( B ) Age-related splicing changes in the protocadherin gamma transcript in the human PFC and CBC. RNA-seq coverage in all 12 DS1 samples is shown. ( C) Inclusion ratio profiles of the protocadherin gamma C 3 -C 5 exons. The colors represent the RNA-seq data sets analyzed: yellow—DS1/PFC, gray—DS1/CBC, and red—DS2/PFC. The points represent the inclusion ratios and the lines represent the cubic spline curves fitted with three degrees of freedom.

Techniques Used: RNA Sequencing Assay

30) Product Images from "Translational contributions to tissue specificity in rhythmic and constitutive gene expression"

Article Title: Translational contributions to tissue specificity in rhythmic and constitutive gene expression

Journal: Genome Biology

doi: 10.1186/s13059-017-1222-2

Tissue specificity in core clock gene expression at the level of RNA abundance and translation. a Scatterplot of transcript abundance (RNA-seq) vs. footprint abundance (RPF-seq) for liver ( grey ) and kidney ( sepia ) ( n = 10,289), where core clock components are highlighted (kidney, dots with dashed circles ). Coloured dashed lines join the relative locations of each core clock gene between organs. b Bar graph of the average RPKM ratio between kidney and liver for the main circadian core clock genes, at the level of mRNA abundance ( dark shades ) and ribosome footprints ( light shades ) suggested that translational compensation led to higher similarity at the level of protein biosynthesis (RPF) for several core clock genes. c Hierarchical clustering of the organs’ RNA and RPF profiles based on the similarities of the core clock genes expression patterns ( n = 12, genes shown in B). The height of the branches represents weighted average distances over the considered genes (see ‘ Methods ’). Note that RPF rhythms in two organs were more similar than RNA and RPF rhythms within an organ. d Hierarchical clustering as in ( c ) based on the genes detected as rhythmic throughout ( n = 178, see Fig. 3c ). When compared to the clustering based on core clock gene expression patterns in ( c ), this rhythmic gene set showed an organ-based clustering. e Scatterplot of kidney/liver ratios of uORF vs. CDS translation efficiencies for genes containing AUG-initiated translated uORFs in both organs ( n = 1199). uORF-containing core clock genes are highlighted. As also shown in Additional file 1 : Figure S8E, differential uORF usage could not globally explain differences in CDS TE across organs (note the lack of negative correlation between the two variables, R 2 = 0.005, p = 0.008). As an exception, the lower uORF TE of Nr1d2 might have a role in setting relatively higher CDS TE in kidney. f RPF ( blue ) and RNA ( orange ) reads mapping along the Nr1d2 transcript in kidney ( top ) and liver ( bottom ) for the timepoint of maximal CDS translation (ZT10). 5′ UTR and CDS are shown in full, but for better visualization only a portion of the 3′ UTR (the same length as the 5′ UTR) is shown. Red boxes indicate the predicted AUG-initiated translated uORFs. Right panels show that, similar to the CDS, the uORFs showed clear frame preference, indicative of active translation
Figure Legend Snippet: Tissue specificity in core clock gene expression at the level of RNA abundance and translation. a Scatterplot of transcript abundance (RNA-seq) vs. footprint abundance (RPF-seq) for liver ( grey ) and kidney ( sepia ) ( n = 10,289), where core clock components are highlighted (kidney, dots with dashed circles ). Coloured dashed lines join the relative locations of each core clock gene between organs. b Bar graph of the average RPKM ratio between kidney and liver for the main circadian core clock genes, at the level of mRNA abundance ( dark shades ) and ribosome footprints ( light shades ) suggested that translational compensation led to higher similarity at the level of protein biosynthesis (RPF) for several core clock genes. c Hierarchical clustering of the organs’ RNA and RPF profiles based on the similarities of the core clock genes expression patterns ( n = 12, genes shown in B). The height of the branches represents weighted average distances over the considered genes (see ‘ Methods ’). Note that RPF rhythms in two organs were more similar than RNA and RPF rhythms within an organ. d Hierarchical clustering as in ( c ) based on the genes detected as rhythmic throughout ( n = 178, see Fig. 3c ). When compared to the clustering based on core clock gene expression patterns in ( c ), this rhythmic gene set showed an organ-based clustering. e Scatterplot of kidney/liver ratios of uORF vs. CDS translation efficiencies for genes containing AUG-initiated translated uORFs in both organs ( n = 1199). uORF-containing core clock genes are highlighted. As also shown in Additional file 1 : Figure S8E, differential uORF usage could not globally explain differences in CDS TE across organs (note the lack of negative correlation between the two variables, R 2 = 0.005, p = 0.008). As an exception, the lower uORF TE of Nr1d2 might have a role in setting relatively higher CDS TE in kidney. f RPF ( blue ) and RNA ( orange ) reads mapping along the Nr1d2 transcript in kidney ( top ) and liver ( bottom ) for the timepoint of maximal CDS translation (ZT10). 5′ UTR and CDS are shown in full, but for better visualization only a portion of the 3′ UTR (the same length as the 5′ UTR) is shown. Red boxes indicate the predicted AUG-initiated translated uORFs. Right panels show that, similar to the CDS, the uORFs showed clear frame preference, indicative of active translation

Techniques Used: Expressing, RNA Sequencing Assay

Ribosome profiling around-the-clock in mouse liver and kidney. a Overview of the experimental design. Livers and kidneys for ribosome profiling were collected every 2 h for two daily cycles. Each timepoint sample was a pool of organs from two animals. Mice were kept under 12 h:12 h light-dark conditions, with Zeitgeber times ZT00 corresponding to lights-on and ZT12 to lights-off. b Read distribution to transcript features. RPF-seq ( left ; kidney in orange , liver in green ) and RNA sequencing (RNA-seq) ( right ; blue and red for kidney and liver, respectively) compared with a distribution expected from the relative feature sizes ( grey ; the distributions based on feature sizes were highly similar for both organs, thus only that for kidney is shown). Note that RPF-seq footprints were enriched on the CDS and depleted from UTRs, whereas RNA-seq reads distributed more homogeneously along transcripts, according to feature size. Of note, the higher level of 3′ UTR footprints in kidney resulted mainly from differences in the efficiency with which stop codon footprints were captured, as described in ( c ). c Predicted position of the ribosome’s aminoacyl tRNA-site (A-site) of reads relative to the CDS start and stop codons. Read density at each position was averaged across single protein coding isoform genes (i.e., genes with one main expressed transcript isoform) that had an average RPF RPKM > 5, a CDS > 400 nt in length and were expressed in both organs ( n = 3037 genes). This analysis revealed the trinucleotide periodicity of RPF-seq (but not RNA-seq) reads in both organs. Inset : frame analysis of CDS reads showed preference for the annotated reading frame (frame 1, the same frame as the start codon) in RPF but not in RNA reads. Violin plots extend to the range of the data ( n = 3694 genes for liver, n = 4602 genes for kidney). A separate analysis of the higher level of stop codon footprints in kidney, that also led to the differences in 3′ UTR reads in B, can be found in Additional file 1 : Figure S2A, B. d Principal component analysis (PCA) of kidney and liver RPF-seq and RNA-seq datasets, using the 4000 most variable genes. The first two components reflected the variability coming from organ (PC1, 64.21%) and from RPF/RNA origin of datasets (PC2, 28.35%). e PC3 vs. PC5 (together 12.5% of variation) resolved the factor time within each dataset, leading to a representation that resembled the face of a clock. Each dot represents one sample, timepoint replicates are joined by a line and timepoints within each dataset are sequentially coloured . The circular arrangement was larger for liver than kidney, suggesting a higher contribution of hepatic rhythmic genes to overall variability. Additional file 1 : Figure S4 shows the scree plot for the ten first components
Figure Legend Snippet: Ribosome profiling around-the-clock in mouse liver and kidney. a Overview of the experimental design. Livers and kidneys for ribosome profiling were collected every 2 h for two daily cycles. Each timepoint sample was a pool of organs from two animals. Mice were kept under 12 h:12 h light-dark conditions, with Zeitgeber times ZT00 corresponding to lights-on and ZT12 to lights-off. b Read distribution to transcript features. RPF-seq ( left ; kidney in orange , liver in green ) and RNA sequencing (RNA-seq) ( right ; blue and red for kidney and liver, respectively) compared with a distribution expected from the relative feature sizes ( grey ; the distributions based on feature sizes were highly similar for both organs, thus only that for kidney is shown). Note that RPF-seq footprints were enriched on the CDS and depleted from UTRs, whereas RNA-seq reads distributed more homogeneously along transcripts, according to feature size. Of note, the higher level of 3′ UTR footprints in kidney resulted mainly from differences in the efficiency with which stop codon footprints were captured, as described in ( c ). c Predicted position of the ribosome’s aminoacyl tRNA-site (A-site) of reads relative to the CDS start and stop codons. Read density at each position was averaged across single protein coding isoform genes (i.e., genes with one main expressed transcript isoform) that had an average RPF RPKM > 5, a CDS > 400 nt in length and were expressed in both organs ( n = 3037 genes). This analysis revealed the trinucleotide periodicity of RPF-seq (but not RNA-seq) reads in both organs. Inset : frame analysis of CDS reads showed preference for the annotated reading frame (frame 1, the same frame as the start codon) in RPF but not in RNA reads. Violin plots extend to the range of the data ( n = 3694 genes for liver, n = 4602 genes for kidney). A separate analysis of the higher level of stop codon footprints in kidney, that also led to the differences in 3′ UTR reads in B, can be found in Additional file 1 : Figure S2A, B. d Principal component analysis (PCA) of kidney and liver RPF-seq and RNA-seq datasets, using the 4000 most variable genes. The first two components reflected the variability coming from organ (PC1, 64.21%) and from RPF/RNA origin of datasets (PC2, 28.35%). e PC3 vs. PC5 (together 12.5% of variation) resolved the factor time within each dataset, leading to a representation that resembled the face of a clock. Each dot represents one sample, timepoint replicates are joined by a line and timepoints within each dataset are sequentially coloured . The circular arrangement was larger for liver than kidney, suggesting a higher contribution of hepatic rhythmic genes to overall variability. Additional file 1 : Figure S4 shows the scree plot for the ten first components

Techniques Used: Mouse Assay, RNA Sequencing Assay

Cross-organ differences in translation efficiency partially compensate RNA abundance differences and show association with transcript features. a Venn diagram showing the gene expression overlap (i.e. genes detected at both RPF and RNA level) between kidney ( yellow , n = 12,423 genes) and liver ( green , n = 10,676 genes). Same cutoffs on RPKM (reads per kilobase of transcript per million mapped reads) were used for both organs. b Scatterplot of kidney-to-liver ratio of mRNA abundance versus translation efficiency (TE) for all expressed genes ( n = 10,289), averaged over all timepoints. Corresponding density curves are plotted on the margins. Dashed red lines represent the 2.5 and 97.5 percentiles of each variable and the corresponding fold-change is indicated. Linear regression line is depicted in blue (R 2 = 0.0009, p = 0.0009). While 95% of genes spanned a 114-fold range in mRNA abundance differences across organs, the same number of genes changed less than threefold in TE, underlining that transcript abundance was the main contributor to divergent gene expression. c Inter-organ Spearman correlation for RNA-seq and RPF-seq samples. Each dot represents the correlation coefficient between kidney and liver for a timepoint and replicate sample. Note that RPF-seq samples consistently correlated significantly better than RNA-seq samples ( p
Figure Legend Snippet: Cross-organ differences in translation efficiency partially compensate RNA abundance differences and show association with transcript features. a Venn diagram showing the gene expression overlap (i.e. genes detected at both RPF and RNA level) between kidney ( yellow , n = 12,423 genes) and liver ( green , n = 10,676 genes). Same cutoffs on RPKM (reads per kilobase of transcript per million mapped reads) were used for both organs. b Scatterplot of kidney-to-liver ratio of mRNA abundance versus translation efficiency (TE) for all expressed genes ( n = 10,289), averaged over all timepoints. Corresponding density curves are plotted on the margins. Dashed red lines represent the 2.5 and 97.5 percentiles of each variable and the corresponding fold-change is indicated. Linear regression line is depicted in blue (R 2 = 0.0009, p = 0.0009). While 95% of genes spanned a 114-fold range in mRNA abundance differences across organs, the same number of genes changed less than threefold in TE, underlining that transcript abundance was the main contributor to divergent gene expression. c Inter-organ Spearman correlation for RNA-seq and RPF-seq samples. Each dot represents the correlation coefficient between kidney and liver for a timepoint and replicate sample. Note that RPF-seq samples consistently correlated significantly better than RNA-seq samples ( p

Techniques Used: Expressing, RNA Sequencing Assay

High tissue divergence in translationally driven rhythms. a Venn diagram of rhythmic RPF-seq sets in kidney ( yellow , n = 92) and liver ( green , n = 142) after the Babel analysis indicated strong tissue specificity of translational control. b Daily profiles of RPF-seq RPKM ( blue ) and RNA-seq RPKM ( orange ) for the two genes detected as translationally regulated in both tissues in ( a ). c , d Circular phase histogram for the 92 ( c , kidney) and 142 ( d , liver) genes showing footprint rhythmicity in the organs. Note that the translational upregulation of transcripts observed at the day-to-night transition in liver was absent in kidney. e , f Heatmaps of RNA ( left panels ) and RPF ( right panels ) rhythms for the 92 and 142 genes translationally regulated in kidney ( e ) and in liver ( f ), respectively. Genes are sorted by footprint phase and expression levels are standardized by row (gene). These sets of genes showed rhythmicity in footprint abundance but no oscillation in mRNA. g , h Daily profiles of RPF-seq RPKM ( blue ) and RNA-seq RPKM ( orange ) for representative examples of translationally generated rhythms specific for liver ( g ) and kidney ( h ). For each gene, the upper panel shows the kidney data and the lower panel the liver data. Hoxd3 was not expressed in liver. i Translation efficiency (TE) around-the-clock for ribosomal protein (RP) genes expressed in liver ( green , n = 86) and in kidney ( yellow , n = 89). For each timepoint (ZT) boxplots represent the interquartile range and whiskers extend to the minimum and maximum TE within 1.5 times the interquantile range. Lines connect the median of each boxplot to ease visualization. Note the global TE upregulation at ZT10 in liver, whereas TEs in kidney remain high over the day
Figure Legend Snippet: High tissue divergence in translationally driven rhythms. a Venn diagram of rhythmic RPF-seq sets in kidney ( yellow , n = 92) and liver ( green , n = 142) after the Babel analysis indicated strong tissue specificity of translational control. b Daily profiles of RPF-seq RPKM ( blue ) and RNA-seq RPKM ( orange ) for the two genes detected as translationally regulated in both tissues in ( a ). c , d Circular phase histogram for the 92 ( c , kidney) and 142 ( d , liver) genes showing footprint rhythmicity in the organs. Note that the translational upregulation of transcripts observed at the day-to-night transition in liver was absent in kidney. e , f Heatmaps of RNA ( left panels ) and RPF ( right panels ) rhythms for the 92 and 142 genes translationally regulated in kidney ( e ) and in liver ( f ), respectively. Genes are sorted by footprint phase and expression levels are standardized by row (gene). These sets of genes showed rhythmicity in footprint abundance but no oscillation in mRNA. g , h Daily profiles of RPF-seq RPKM ( blue ) and RNA-seq RPKM ( orange ) for representative examples of translationally generated rhythms specific for liver ( g ) and kidney ( h ). For each gene, the upper panel shows the kidney data and the lower panel the liver data. Hoxd3 was not expressed in liver. i Translation efficiency (TE) around-the-clock for ribosomal protein (RP) genes expressed in liver ( green , n = 86) and in kidney ( yellow , n = 89). For each timepoint (ZT) boxplots represent the interquartile range and whiskers extend to the minimum and maximum TE within 1.5 times the interquantile range. Lines connect the median of each boxplot to ease visualization. Note the global TE upregulation at ZT10 in liver, whereas TEs in kidney remain high over the day

Techniques Used: RNA Sequencing Assay, Expressing, Generated

Rhythmicity analyses across organs reveals phase modulation by translation in kidney. a Venn diagram showing rhythmic genes in kidney. Of the 12,423 expressed genes, 1338 showed 24-h oscillations of > 1.5-fold amplitude in mRNA abundance (RNA-seq, 10.7%) and 977 in footprint abundance (RPF-seq, 7.9%). A total of 542 genes (4.3%) were identified as rhythmic at both levels. b Cumulative distribution of phase differences (RPF peak – RNA peak, in hours) for genes rhythmic at both RNA-seq and RPF-seq in liver ( green , n = 1178) and kidney ( yellow , n = 542). The two distributions were significantly different ( p
Figure Legend Snippet: Rhythmicity analyses across organs reveals phase modulation by translation in kidney. a Venn diagram showing rhythmic genes in kidney. Of the 12,423 expressed genes, 1338 showed 24-h oscillations of > 1.5-fold amplitude in mRNA abundance (RNA-seq, 10.7%) and 977 in footprint abundance (RPF-seq, 7.9%). A total of 542 genes (4.3%) were identified as rhythmic at both levels. b Cumulative distribution of phase differences (RPF peak – RNA peak, in hours) for genes rhythmic at both RNA-seq and RPF-seq in liver ( green , n = 1178) and kidney ( yellow , n = 542). The two distributions were significantly different ( p

Techniques Used: RNA Sequencing Assay

Related Articles

Clone Assay:

Article Title: Correlation of LNCR rasiRNAs Expression with Heterochromatin Formation during Development of the Holocentric Insect Spodoptera frugiperda
Article Snippet: .. After elimination of redundancy, sequences were mapped to the available genomic sequences cloned in 37 BACs (1% of S. frugiperda's genome), RNA libraries and the annotated DNA repeated elements using BLASTn, allowing no mismatch (100% of sequence identity all along their length). .. The sequences of small RNAs with 100% of sequence identity all along their length were used for further analysis.

Amplification:

Article Title: Distinct Expression Profiles and Novel Targets of MicroRNAs in Human Spermatogonia, Pachytene Spermatocytes, and Round Spermatids between OA Patients and NOA Patients
Article Snippet: .. The cDNA libraries of small RNAs were established using 100 ng of total RNA, and adaptor ligation, first-strand cDNA synthesis, PCR amplification, gel purification, and sequencing of small RNA libraries using an Illumina HiSeq 2000 were performed according to the procedure as described previously. .. Further analyses of small RNA expression and mapping of microRNAs, piRNA, endo-siRNA, and other small RNAs were conducted pursuant to the previous method.

Construct:

Article Title: Efficiency and precision of microRNA biogenesis modes in plants
Article Snippet: .. The small RNA library was constructed using the Illumina TruSeq sRNA kit, and sequenced on the Illumina HiSeq platform at the University of Delaware. ..

Article Title: Human MicroRNA miR-532-5p Exhibits Antiviral Activity against West Nile Virus via Suppression of Host Genes SESTD1 and TAB3 Required for Virus Replication
Article Snippet: Enriched small-RNA fractions isolated from infected and uninfected HEK293 cells were used to construct libraries of mature miRNAs and pre-miRNAs. .. The RNA library for pre-miRNA sequencing was prepared using an mRNA-Seq Sample Prep kit (Illumina, USA), and sequencing was conducted on an Illumina HiSeq 2000 instrument according to the manufacturer's protocol.

Article Title: Characterization of Transcriptional Complexity during Adipose Tissue Development in Bovines of Different Ages and Sexes
Article Snippet: .. RNA sequencing of bovine subcutaneous adipose tissues To obtain a global view of the bovine adipose tissue transcriptome, total RNA from the subcutaneous adipose tissues of fetal bovines, adult bulls, adult heifers and adult steers (ATFB, ATAB, ATAH and ATAS) were used to construct RNA libraries for Illumina sequencing. ..

Real-time Polymerase Chain Reaction:

Article Title: miRNA alteration is an important mechanism in sugarcane response to low-temperature environment
Article Snippet: .. The stem loop primers of miRNAs in sugarcane (DOCX 14 kb) The real time PCR primers of miRNAs and target gene in sugarcane (DOCX 15 kb) The known and novel miRNAs in 4 small RNA libraries (F0, F3, R0 and R3) (XLSX 30 kb) Prediction of target genes of the known and novel miRNAs (XLSX 208 kb) GO analysis of the predicted targets of known and novel miRNAs (XLSX 243 kb) KEGG analysis of the predicted targets of known miRNAs in F3/F0 (XLSX 12 kb) KEGG analysis of the predicted targets of known miRNAs in R3/R0 (XLSX 11 kb) KEGG analysis of the predicted targets of novel miRNAs in F3/F0 (XLSX 15 kb) KEGG analysis of the predicted targets of novel miRNAs in R3/R0 (XLSX 12 kb) The stem longitudinal section of sugarcane FN39 (relative cold tolerance) and ROC22 (relative cold sensitivity) in plant tip part after cold stress, indicating that more serious damage was observed in ROC22 in its stem tissues and growing point (TIFF 31806 kb) Validation of the cold-stress treatment in sugarcane. .. The expression patterns of three cold responsive genes, CBF1 , CBF3 and NAC23 , were detected by RT-qPCR in sugarcane FN39 (A) and ROC22 (B) cultivars.

Microarray:

Article Title: Chicken interferome: avian interferon-stimulated genes identified by microarray and RNA-seq of primary chick embryo fibroblasts treated with a chicken type I interferon (IFN-α)
Article Snippet: The cell samples used were identical to those used for the microarray analyses. .. Total RNA was extracted as for microarrays (above) and RNA libraries were prepared for deep sequencing using the TruSeq RNA Sample Preparation Kit (Illumina) according to the manufacturer’s instructions.

Incubation:

Article Title: RNA-Seq analyses reveal the order of tRNA processing events and the maturation of C/D box and CRISPR RNAs in the hyperthermophile Methanopyrus kandleri
Article Snippet: Subsequently, 2 mM adenosine triphosphate (ATP) and 10 U T4 PNK were added, and the reaction mixture was incubated for 1 h at 37°C to generate monophosphorylated 5′ termini. .. RNA libraries were prepared with an Illumina TruSeq RNA Sample Prep Kit (Ambion), and sequencing on an Illumina HiSeq2000 sequencer was performed at the Max-Planck Genomecentre, Cologne (Max Planck Institute for Plant Breeding Research, Köln, Germany).

Expressing:

Article Title: miRNA alteration is an important mechanism in sugarcane response to low-temperature environment
Article Snippet: In this study, a total of 412 sugarcane miRNAs, including 261 known and 151 novel miRNAs, were obtained from 4 small RNA libraries through the Illumina sequencing method. .. Among them, 62 exhibited significant differential expression under cold stress, with 34 being upregulated and 28 being downregulated.

Article Title: Genetic Predisposition for Beta Cell Fragility Underlies Type 1 and Type 2 Diabetes
Article Snippet: An RNA library was prepared with TruSeq stranded mRNA library prep (Illumina), and RNA-seq was performed using the HiSeq 2000 300-Gb flow cell. .. For assessment of insHEL expression, reads were aligned to a custom genome build combining the Ensembl build of chicken chromosome 1, containing LYZ (ENSGALG00000009963) with its corresponding transcript ENSGALT00000016916, with the mouse genome (Ensembl builds Galgal4.75 and GRCm38.75, respectively) using TopHat v 2.0.11.

Article Title: Characterization of Transcriptional Complexity during Adipose Tissue Development in Bovines of Different Ages and Sexes
Article Snippet: RNA sequencing of bovine subcutaneous adipose tissues To obtain a global view of the bovine adipose tissue transcriptome, total RNA from the subcutaneous adipose tissues of fetal bovines, adult bulls, adult heifers and adult steers (ATFB, ATAB, ATAH and ATAS) were used to construct RNA libraries for Illumina sequencing. .. Gene expression level was calculated using the reads per kilobase transcriptome per million mapped reads (RPKM) method.

Derivative Assay:

Article Title: Deep mRNA sequencing reveals stage-specific transcriptome alterations during microsclerotia development in the smoke tree vascular wilt pathogen, Verticillium dahliae
Article Snippet: .. Overview of the V. dahliae transcriptome The pipeline of RNA-Seq analysis is shown in Figure B, RNA libraries derived from the samples were pair-end sequenced using an Illumina high-throughput sequencing platform. ..

Gel Purification:

Article Title: Distinct Expression Profiles and Novel Targets of MicroRNAs in Human Spermatogonia, Pachytene Spermatocytes, and Round Spermatids between OA Patients and NOA Patients
Article Snippet: .. The cDNA libraries of small RNAs were established using 100 ng of total RNA, and adaptor ligation, first-strand cDNA synthesis, PCR amplification, gel purification, and sequencing of small RNA libraries using an Illumina HiSeq 2000 were performed according to the procedure as described previously. .. Further analyses of small RNA expression and mapping of microRNAs, piRNA, endo-siRNA, and other small RNAs were conducted pursuant to the previous method.

Flow Cytometry:

Article Title: Genetic Predisposition for Beta Cell Fragility Underlies Type 1 and Type 2 Diabetes
Article Snippet: .. An RNA library was prepared with TruSeq stranded mRNA library prep (Illumina), and RNA-seq was performed using the HiSeq 2000 300-Gb flow cell. .. Sequence reads were mapped to the mouse reference genome (assembly GRCm38.73) using TopHat v2.0.8b .

Ligation:

Article Title: Distinct Expression Profiles and Novel Targets of MicroRNAs in Human Spermatogonia, Pachytene Spermatocytes, and Round Spermatids between OA Patients and NOA Patients
Article Snippet: .. The cDNA libraries of small RNAs were established using 100 ng of total RNA, and adaptor ligation, first-strand cDNA synthesis, PCR amplification, gel purification, and sequencing of small RNA libraries using an Illumina HiSeq 2000 were performed according to the procedure as described previously. .. Further analyses of small RNA expression and mapping of microRNAs, piRNA, endo-siRNA, and other small RNAs were conducted pursuant to the previous method.

Genomic Sequencing:

Article Title: Correlation of LNCR rasiRNAs Expression with Heterochromatin Formation during Development of the Holocentric Insect Spodoptera frugiperda
Article Snippet: .. After elimination of redundancy, sequences were mapped to the available genomic sequences cloned in 37 BACs (1% of S. frugiperda's genome), RNA libraries and the annotated DNA repeated elements using BLASTn, allowing no mismatch (100% of sequence identity all along their length). .. The sequences of small RNAs with 100% of sequence identity all along their length were used for further analysis.

Infection:

Article Title: Human MicroRNA miR-532-5p Exhibits Antiviral Activity against West Nile Virus via Suppression of Host Genes SESTD1 and TAB3 Required for Virus Replication
Article Snippet: Enriched small-RNA fractions isolated from infected and uninfected HEK293 cells were used to construct libraries of mature miRNAs and pre-miRNAs. .. The RNA library for pre-miRNA sequencing was prepared using an mRNA-Seq Sample Prep kit (Illumina, USA), and sequencing was conducted on an Illumina HiSeq 2000 instrument according to the manufacturer's protocol.

Generated:

Article Title: Human MicroRNA miR-532-5p Exhibits Antiviral Activity against West Nile Virus via Suppression of Host Genes SESTD1 and TAB3 Required for Virus Replication
Article Snippet: A mature miRNA library was generated using a Truseq small-RNA preparation kit (Illumina, USA) according to Illumina's sample preparation guide. .. The RNA library for pre-miRNA sequencing was prepared using an mRNA-Seq Sample Prep kit (Illumina, USA), and sequencing was conducted on an Illumina HiSeq 2000 instrument according to the manufacturer's protocol.

Sequencing:

Article Title: Chicken interferome: avian interferon-stimulated genes identified by microarray and RNA-seq of primary chick embryo fibroblasts treated with a chicken type I interferon (IFN-α)
Article Snippet: .. Total RNA was extracted as for microarrays (above) and RNA libraries were prepared for deep sequencing using the TruSeq RNA Sample Preparation Kit (Illumina) according to the manufacturer’s instructions. ..

Article Title: Distinct Expression Profiles and Novel Targets of MicroRNAs in Human Spermatogonia, Pachytene Spermatocytes, and Round Spermatids between OA Patients and NOA Patients
Article Snippet: .. The cDNA libraries of small RNAs were established using 100 ng of total RNA, and adaptor ligation, first-strand cDNA synthesis, PCR amplification, gel purification, and sequencing of small RNA libraries using an Illumina HiSeq 2000 were performed according to the procedure as described previously. .. Further analyses of small RNA expression and mapping of microRNAs, piRNA, endo-siRNA, and other small RNAs were conducted pursuant to the previous method.

Article Title: The 25–26 nt Small RNAs in Phytophthora parasitica Are Associated with Efficient Silencing of Homologous Endogenous Genes
Article Snippet: Paragraph title: RNA Isolation, Library Construction and High-Throughput Sequencing ... Briefly, sRNA libraries and RNA libraries were prepared using 1 μg of total RNA according to the protocols of TruSeq Small RNA Library Prep Kit and TruSeq RNA Library Prep Kit (Illumina, San Diego, CA, USA), respectively, and then sequenced on an Illumina Hiseq2500 machine.

Article Title: miRNA alteration is an important mechanism in sugarcane response to low-temperature environment
Article Snippet: .. In this study, a total of 412 sugarcane miRNAs, including 261 known and 151 novel miRNAs, were obtained from 4 small RNA libraries through the Illumina sequencing method. .. Among them, 62 exhibited significant differential expression under cold stress, with 34 being upregulated and 28 being downregulated.

Article Title: Genetic Predisposition for Beta Cell Fragility Underlies Type 1 and Type 2 Diabetes
Article Snippet: An RNA library was prepared with TruSeq stranded mRNA library prep (Illumina), and RNA-seq was performed using the HiSeq 2000 300-Gb flow cell. .. Sequence reads were mapped to the mouse reference genome (assembly GRCm38.73) using TopHat v2.0.8b .

Article Title: Human MicroRNA miR-532-5p Exhibits Antiviral Activity against West Nile Virus via Suppression of Host Genes SESTD1 and TAB3 Required for Virus Replication
Article Snippet: .. The RNA library for pre-miRNA sequencing was prepared using an mRNA-Seq Sample Prep kit (Illumina, USA), and sequencing was conducted on an Illumina HiSeq 2000 instrument according to the manufacturer's protocol. ..

Article Title: Characterization of Transcriptional Complexity during Adipose Tissue Development in Bovines of Different Ages and Sexes
Article Snippet: .. RNA sequencing of bovine subcutaneous adipose tissues To obtain a global view of the bovine adipose tissue transcriptome, total RNA from the subcutaneous adipose tissues of fetal bovines, adult bulls, adult heifers and adult steers (ATFB, ATAB, ATAH and ATAS) were used to construct RNA libraries for Illumina sequencing. ..

Article Title: Deep mRNA sequencing reveals stage-specific transcriptome alterations during microsclerotia development in the smoke tree vascular wilt pathogen, Verticillium dahliae
Article Snippet: .. Overview of the V. dahliae transcriptome The pipeline of RNA-Seq analysis is shown in Figure B, RNA libraries derived from the samples were pair-end sequenced using an Illumina high-throughput sequencing platform. ..

Article Title: Sendai Virus Infection Induces Expression of Novel RNAs in Human Cells
Article Snippet: .. Duplicate samples of mock-infected and Sendai virus-infected cells for 6 hours were used to prepare RNA libraries for sequencing on Illumina HiSeq2000 platform (Illumina) to generate 100 bp paired-end sequencing reads. ..

Article Title: RNA-Seq analyses reveal the order of tRNA processing events and the maturation of C/D box and CRISPR RNAs in the hyperthermophile Methanopyrus kandleri
Article Snippet: .. RNA libraries were prepared with an Illumina TruSeq RNA Sample Prep Kit (Ambion), and sequencing on an Illumina HiSeq2000 sequencer was performed at the Max-Planck Genomecentre, Cologne (Max Planck Institute for Plant Breeding Research, Köln, Germany). .. Identification of small RNA species Sequencing reads were trimmed by (i) removal of Illumina TruSeq linkers and poly-A tails and (ii) removal of sequences using a quality score limit of 0.05.

Article Title: Correlation of LNCR rasiRNAs Expression with Heterochromatin Formation during Development of the Holocentric Insect Spodoptera frugiperda
Article Snippet: .. After elimination of redundancy, sequences were mapped to the available genomic sequences cloned in 37 BACs (1% of S. frugiperda's genome), RNA libraries and the annotated DNA repeated elements using BLASTn, allowing no mismatch (100% of sequence identity all along their length). .. The sequences of small RNAs with 100% of sequence identity all along their length were used for further analysis.

Multiplexing:

Article Title: Chicken interferome: avian interferon-stimulated genes identified by microarray and RNA-seq of primary chick embryo fibroblasts treated with a chicken type I interferon (IFN-α)
Article Snippet: Total RNA was extracted as for microarrays (above) and RNA libraries were prepared for deep sequencing using the TruSeq RNA Sample Preparation Kit (Illumina) according to the manufacturer’s instructions. .. A total of six RNA adapter indices were randomly assigned to the 12 samples to allow multiplexing of libraries.

RNA Sequencing Assay:

Article Title: Chicken interferome: avian interferon-stimulated genes identified by microarray and RNA-seq of primary chick embryo fibroblasts treated with a chicken type I interferon (IFN-α)
Article Snippet: Paragraph title: RNA-seq ... Total RNA was extracted as for microarrays (above) and RNA libraries were prepared for deep sequencing using the TruSeq RNA Sample Preparation Kit (Illumina) according to the manufacturer’s instructions.

Article Title: Genetic Predisposition for Beta Cell Fragility Underlies Type 1 and Type 2 Diabetes
Article Snippet: .. An RNA library was prepared with TruSeq stranded mRNA library prep (Illumina), and RNA-seq was performed using the HiSeq 2000 300-Gb flow cell. .. Sequence reads were mapped to the mouse reference genome (assembly GRCm38.73) using TopHat v2.0.8b .

Article Title: Characterization of Transcriptional Complexity during Adipose Tissue Development in Bovines of Different Ages and Sexes
Article Snippet: .. RNA sequencing of bovine subcutaneous adipose tissues To obtain a global view of the bovine adipose tissue transcriptome, total RNA from the subcutaneous adipose tissues of fetal bovines, adult bulls, adult heifers and adult steers (ATFB, ATAB, ATAH and ATAS) were used to construct RNA libraries for Illumina sequencing. ..

Article Title: Deep mRNA sequencing reveals stage-specific transcriptome alterations during microsclerotia development in the smoke tree vascular wilt pathogen, Verticillium dahliae
Article Snippet: .. Overview of the V. dahliae transcriptome The pipeline of RNA-Seq analysis is shown in Figure B, RNA libraries derived from the samples were pair-end sequenced using an Illumina high-throughput sequencing platform. ..

Article Title: Sendai Virus Infection Induces Expression of Novel RNAs in Human Cells
Article Snippet: Paragraph title: RNA sequencing and transcript assembly ... Duplicate samples of mock-infected and Sendai virus-infected cells for 6 hours were used to prepare RNA libraries for sequencing on Illumina HiSeq2000 platform (Illumina) to generate 100 bp paired-end sequencing reads.

Article Title: RNA-Seq analyses reveal the order of tRNA processing events and the maturation of C/D box and CRISPR RNAs in the hyperthermophile Methanopyrus kandleri
Article Snippet: Samples 4 to 6 resulted in highly similar RNA-Seq sequencing output. .. RNA libraries were prepared with an Illumina TruSeq RNA Sample Prep Kit (Ambion), and sequencing on an Illumina HiSeq2000 sequencer was performed at the Max-Planck Genomecentre, Cologne (Max Planck Institute for Plant Breeding Research, Köln, Germany).

Article Title: Human PRPF40B regulates hundreds of alternative splicing targets and represses a hypoxia expression signature
Article Snippet: Paragraph title: RNA-sequencing library preparation ... We prepared RNA libraries using the TruSeq Stranded mRNA Library Prep Kit (Illumina).

Magnetic Beads:

Article Title: Human PRPF40B regulates hundreds of alternative splicing targets and represses a hypoxia expression signature
Article Snippet: We prepared RNA libraries using the TruSeq Stranded mRNA Library Prep Kit (Illumina). .. In brief, we purified poly(A) containing mRNAs by poly(T) oligonucleotides attached to magnetic beads.

Isolation:

Article Title: The 25–26 nt Small RNAs in Phytophthora parasitica Are Associated with Efficient Silencing of Homologous Endogenous Genes
Article Snippet: Paragraph title: RNA Isolation, Library Construction and High-Throughput Sequencing ... Briefly, sRNA libraries and RNA libraries were prepared using 1 μg of total RNA according to the protocols of TruSeq Small RNA Library Prep Kit and TruSeq RNA Library Prep Kit (Illumina, San Diego, CA, USA), respectively, and then sequenced on an Illumina Hiseq2500 machine.

Article Title: Genetic Predisposition for Beta Cell Fragility Underlies Type 1 and Type 2 Diabetes
Article Snippet: Total RNA was prepared from isolated islets by RNeasy Mini kit (Qiagen). .. An RNA library was prepared with TruSeq stranded mRNA library prep (Illumina), and RNA-seq was performed using the HiSeq 2000 300-Gb flow cell.

Article Title: Human MicroRNA miR-532-5p Exhibits Antiviral Activity against West Nile Virus via Suppression of Host Genes SESTD1 and TAB3 Required for Virus Replication
Article Snippet: Enriched small-RNA fractions isolated from infected and uninfected HEK293 cells were used to construct libraries of mature miRNAs and pre-miRNAs. .. The RNA library for pre-miRNA sequencing was prepared using an mRNA-Seq Sample Prep kit (Illumina, USA), and sequencing was conducted on an Illumina HiSeq 2000 instrument according to the manufacturer's protocol.

Purification:

Article Title: Human MicroRNA miR-532-5p Exhibits Antiviral Activity against West Nile Virus via Suppression of Host Genes SESTD1 and TAB3 Required for Virus Replication
Article Snippet: The purified cDNA library was used for cluster generation on Illumina's Cluster Station and then sequenced on an Illumina GAIIx following the vendor's instructions. .. The RNA library for pre-miRNA sequencing was prepared using an mRNA-Seq Sample Prep kit (Illumina, USA), and sequencing was conducted on an Illumina HiSeq 2000 instrument according to the manufacturer's protocol.

Article Title: Human PRPF40B regulates hundreds of alternative splicing targets and represses a hypoxia expression signature
Article Snippet: We prepared RNA libraries using the TruSeq Stranded mRNA Library Prep Kit (Illumina). .. In brief, we purified poly(A) containing mRNAs by poly(T) oligonucleotides attached to magnetic beads.

Polymerase Chain Reaction:

Article Title: Distinct Expression Profiles and Novel Targets of MicroRNAs in Human Spermatogonia, Pachytene Spermatocytes, and Round Spermatids between OA Patients and NOA Patients
Article Snippet: .. The cDNA libraries of small RNAs were established using 100 ng of total RNA, and adaptor ligation, first-strand cDNA synthesis, PCR amplification, gel purification, and sequencing of small RNA libraries using an Illumina HiSeq 2000 were performed according to the procedure as described previously. .. Further analyses of small RNA expression and mapping of microRNAs, piRNA, endo-siRNA, and other small RNAs were conducted pursuant to the previous method.

Quantitative RT-PCR:

Article Title: miRNA alteration is an important mechanism in sugarcane response to low-temperature environment
Article Snippet: In this study, a total of 412 sugarcane miRNAs, including 261 known and 151 novel miRNAs, were obtained from 4 small RNA libraries through the Illumina sequencing method. .. The expression of 13 miRNAs and 12 corresponding targets was validated by RT-qPCR, with the majority being consistent with the sequencing data.

cDNA Library Assay:

Article Title: Human MicroRNA miR-532-5p Exhibits Antiviral Activity against West Nile Virus via Suppression of Host Genes SESTD1 and TAB3 Required for Virus Replication
Article Snippet: The purified cDNA library was used for cluster generation on Illumina's Cluster Station and then sequenced on an Illumina GAIIx following the vendor's instructions. .. The RNA library for pre-miRNA sequencing was prepared using an mRNA-Seq Sample Prep kit (Illumina, USA), and sequencing was conducted on an Illumina HiSeq 2000 instrument according to the manufacturer's protocol.

Agarose Gel Electrophoresis:

Article Title: A Versatile and Robust Serine Protease Inhibitor Scaffold from Actinia tenebrosa
Article Snippet: RNA was visually inspected on a 1.5% agarose gel stained with GelRed (Biotium). .. RNA libraries were prepared using the TruSeq Stranded mRNA Library Preparation Kit (Illumina, San Diego, CA, USA) as described in the manufacturer’s instructions.

Chromatin Immunoprecipitation:

Article Title: A Versatile and Robust Serine Protease Inhibitor Scaffold from Actinia tenebrosa
Article Snippet: Further quality assessment was performed using an RNA nano chip on a Bioanalyzer 2100 (Agilent) to confirm RNA integrity and concentration. .. RNA libraries were prepared using the TruSeq Stranded mRNA Library Preparation Kit (Illumina, San Diego, CA, USA) as described in the manufacturer’s instructions.

Article Title: Chicken interferome: avian interferon-stimulated genes identified by microarray and RNA-seq of primary chick embryo fibroblasts treated with a chicken type I interferon (IFN-α)
Article Snippet: Total RNA was extracted as for microarrays (above) and RNA libraries were prepared for deep sequencing using the TruSeq RNA Sample Preparation Kit (Illumina) according to the manufacturer’s instructions. .. At the end of the protocol, libraries were quantified using a Nanodrop Spectrophotometer and checked for quality using a 2100 Bioanalyzer High Sensitivity DNA chip (Agilent Technologies).

Software:

Article Title: A Versatile and Robust Serine Protease Inhibitor Scaffold from Actinia tenebrosa
Article Snippet: RNA libraries were prepared using the TruSeq Stranded mRNA Library Preparation Kit (Illumina, San Diego, CA, USA) as described in the manufacturer’s instructions. .. High-quality reads ( > Q30, < 1% ambiguities) were assembled using the Trinity de novo assembler software version 2.2.0 (Broad Institute, Cambridge, MA, USA) [ ].

Article Title: Human MicroRNA miR-532-5p Exhibits Antiviral Activity against West Nile Virus via Suppression of Host Genes SESTD1 and TAB3 Required for Virus Replication
Article Snippet: Raw sequencing reads (40 nt) were obtained using Illumina's Sequencing Control Studio software version 2.8 (SCS v2.8) following real-time sequencing image analysis and base calling with Illumina's Real-Time Analysis version 1.8.70 (RTA v1.8.70). .. The RNA library for pre-miRNA sequencing was prepared using an mRNA-Seq Sample Prep kit (Illumina, USA), and sequencing was conducted on an Illumina HiSeq 2000 instrument according to the manufacturer's protocol.

RNA Expression:

Article Title: Distinct Expression Profiles and Novel Targets of MicroRNAs in Human Spermatogonia, Pachytene Spermatocytes, and Round Spermatids between OA Patients and NOA Patients
Article Snippet: The cDNA libraries of small RNAs were established using 100 ng of total RNA, and adaptor ligation, first-strand cDNA synthesis, PCR amplification, gel purification, and sequencing of small RNA libraries using an Illumina HiSeq 2000 were performed according to the procedure as described previously. .. Further analyses of small RNA expression and mapping of microRNAs, piRNA, endo-siRNA, and other small RNAs were conducted pursuant to the previous method.

Sample Prep:

Article Title: Chicken interferome: avian interferon-stimulated genes identified by microarray and RNA-seq of primary chick embryo fibroblasts treated with a chicken type I interferon (IFN-α)
Article Snippet: .. Total RNA was extracted as for microarrays (above) and RNA libraries were prepared for deep sequencing using the TruSeq RNA Sample Preparation Kit (Illumina) according to the manufacturer’s instructions. ..

Article Title: Human MicroRNA miR-532-5p Exhibits Antiviral Activity against West Nile Virus via Suppression of Host Genes SESTD1 and TAB3 Required for Virus Replication
Article Snippet: .. The RNA library for pre-miRNA sequencing was prepared using an mRNA-Seq Sample Prep kit (Illumina, USA), and sequencing was conducted on an Illumina HiSeq 2000 instrument according to the manufacturer's protocol. ..

Article Title: RNA-Seq analyses reveal the order of tRNA processing events and the maturation of C/D box and CRISPR RNAs in the hyperthermophile Methanopyrus kandleri
Article Snippet: .. RNA libraries were prepared with an Illumina TruSeq RNA Sample Prep Kit (Ambion), and sequencing on an Illumina HiSeq2000 sequencer was performed at the Max-Planck Genomecentre, Cologne (Max Planck Institute for Plant Breeding Research, Köln, Germany). .. Identification of small RNA species Sequencing reads were trimmed by (i) removal of Illumina TruSeq linkers and poly-A tails and (ii) removal of sequences using a quality score limit of 0.05.

Spectrophotometry:

Article Title: Chicken interferome: avian interferon-stimulated genes identified by microarray and RNA-seq of primary chick embryo fibroblasts treated with a chicken type I interferon (IFN-α)
Article Snippet: Total RNA was extracted as for microarrays (above) and RNA libraries were prepared for deep sequencing using the TruSeq RNA Sample Preparation Kit (Illumina) according to the manufacturer’s instructions. .. At the end of the protocol, libraries were quantified using a Nanodrop Spectrophotometer and checked for quality using a 2100 Bioanalyzer High Sensitivity DNA chip (Agilent Technologies).

Concentration Assay:

Article Title: A Versatile and Robust Serine Protease Inhibitor Scaffold from Actinia tenebrosa
Article Snippet: Further quality assessment was performed using an RNA nano chip on a Bioanalyzer 2100 (Agilent) to confirm RNA integrity and concentration. .. RNA libraries were prepared using the TruSeq Stranded mRNA Library Preparation Kit (Illumina, San Diego, CA, USA) as described in the manufacturer’s instructions.

Article Title: Distinct Expression Profiles and Novel Targets of MicroRNAs in Human Spermatogonia, Pachytene Spermatocytes, and Round Spermatids between OA Patients and NOA Patients
Article Snippet: The concentration and quantity of total RNA were measured by Nanodrop (Thermo), and the ratios of A260 /A280 of total RNA were set as 1.9 to 2.0 to ensure great purity. .. The cDNA libraries of small RNAs were established using 100 ng of total RNA, and adaptor ligation, first-strand cDNA synthesis, PCR amplification, gel purification, and sequencing of small RNA libraries using an Illumina HiSeq 2000 were performed according to the procedure as described previously.

High Throughput Screening Assay:

Article Title: The 25–26 nt Small RNAs in Phytophthora parasitica Are Associated with Efficient Silencing of Homologous Endogenous Genes
Article Snippet: Paragraph title: RNA Isolation, Library Construction and High-Throughput Sequencing ... Briefly, sRNA libraries and RNA libraries were prepared using 1 μg of total RNA according to the protocols of TruSeq Small RNA Library Prep Kit and TruSeq RNA Library Prep Kit (Illumina, San Diego, CA, USA), respectively, and then sequenced on an Illumina Hiseq2500 machine.

Article Title: Deep mRNA sequencing reveals stage-specific transcriptome alterations during microsclerotia development in the smoke tree vascular wilt pathogen, Verticillium dahliae
Article Snippet: .. Overview of the V. dahliae transcriptome The pipeline of RNA-Seq analysis is shown in Figure B, RNA libraries derived from the samples were pair-end sequenced using an Illumina high-throughput sequencing platform. ..

Staining:

Article Title: A Versatile and Robust Serine Protease Inhibitor Scaffold from Actinia tenebrosa
Article Snippet: RNA was visually inspected on a 1.5% agarose gel stained with GelRed (Biotium). .. RNA libraries were prepared using the TruSeq Stranded mRNA Library Preparation Kit (Illumina, San Diego, CA, USA) as described in the manufacturer’s instructions.

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    Illumina Inc traditional rna sequencing library preparation
    Cross-platform comparison of competitive amplicon library preparation with <t>TaqMan</t> qPCR and Illumina <t>RNA-Sequencing.</t> a ) Comparison of TaqMan qPCR with competitive amplicon library preparation (n = 146) for samples A and B without correction for systematic biases. Data is normalized to a median relative abundance. b ) Comparison of Illumina RNA-Sequencing with competitive amplicon library preparation (n = 170) for samples A and B without correction for systematic biases. Data is normalized to a median relative abundance. For a) and b), Spearman’s rank correlation coefficient is noted (r s ). The average of differences for measurements of samples A and B between competitive amplicon library preparation and TaqMan qPCR ( Figure S1 ) or Illumina RNA-sequencing ( Figure S2 ) was determined for each endogenous target; and to illustrate the systematic bias away from the regression line, data points for MMP2 have been highlighted in orange. This difference was subtracted from TaqMan qPCR or Illumina RNA-sequencing measurements for samples C and D and plotted (X-axis). Competitive amplicon library preparation measurements of C and D are plotted on the Y-axis. c) Comparison of TaqMan qPCR with competitive amplicon library preparation (n = 146) for samples C and D with correction for platform and assay specific bias. d) Comparison of Illumina RNA-Sequencing with competitive amplicon library preparation (n = 170) for samples C and D with correction for platform and assay specific bias.
    Traditional Rna Sequencing Library Preparation, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 78/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc bulk rna seq library
    a Comparison of single-cell median and population-level <t>RNA-Seq</t> profiles for cells originating from the <t>U87-exclusive</t> lane in Experiment 1. Each data point was obtained by constructing a median profile from a given number of cells and repeating this 10 times by random sampling with replacement to obtain a median Pearson correlation coefficient and error bar (SEM). This exercise was repeated for comparison to both the U87 and MCF10a bulk RNA-Seq profiles to demonstrate better concordance between the U87 single-cell profiles and the U87 bulk profile. b Same as ( a ), but for single-cell profiles in the MCF10a-exlusive lane. c We conducted differential expression analysis to obtain cell type-specific gene sets for the U87 and MCF10a cells based on single-cell profiles from the pure-cell lanes. Here, we show a histogram of log-ratio of the coefficients of variation (CVs) for the cell type-specific gene sets between the mixed lane profiles and the profiles from the respective pure lanes. As expected, the heterogeneity given by CV is greater for cells in the mixed lanes than in the cell type-exclusive lanes for the cell type-specific genes
    Bulk Rna Seq Library, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 78/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc truseq rna sample preparation kit v2
    Improved comparability across degradation effects and library preparation methods. ( a ) Correlation of unnormalized reads from normally processed iPSC <t>RNA</t> (HICL) and in-process degraded iPSC RNA (HICL_D). Reads for 51,552 loci over the read cutoff of 40 were plotted. ( b ) Correlation of SNV-normalized reads from the HICL and HICL_D samples. ( c ) Agreement of estimates for gene expression in HICL and HICL_D samples estimated by unnormalized reads and SNV-normalized reads. ( d ) Correlation of unnormalized reads generated by <t>TruSeq</t> method (HICL) and NexTera method (HICL_N). Reads for 53,585 loci were plotted. ( e ) Correlation of SNV-normalized reads from the HICL and HICL_N samples. ( f ) Agreement of estimates for gene expression in HICL and HICL_N samples estimated by unnormalized reads and SNV-normalized reads.
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    Cross-platform comparison of competitive amplicon library preparation with TaqMan qPCR and Illumina RNA-Sequencing. a ) Comparison of TaqMan qPCR with competitive amplicon library preparation (n = 146) for samples A and B without correction for systematic biases. Data is normalized to a median relative abundance. b ) Comparison of Illumina RNA-Sequencing with competitive amplicon library preparation (n = 170) for samples A and B without correction for systematic biases. Data is normalized to a median relative abundance. For a) and b), Spearman’s rank correlation coefficient is noted (r s ). The average of differences for measurements of samples A and B between competitive amplicon library preparation and TaqMan qPCR ( Figure S1 ) or Illumina RNA-sequencing ( Figure S2 ) was determined for each endogenous target; and to illustrate the systematic bias away from the regression line, data points for MMP2 have been highlighted in orange. This difference was subtracted from TaqMan qPCR or Illumina RNA-sequencing measurements for samples C and D and plotted (X-axis). Competitive amplicon library preparation measurements of C and D are plotted on the Y-axis. c) Comparison of TaqMan qPCR with competitive amplicon library preparation (n = 146) for samples C and D with correction for platform and assay specific bias. d) Comparison of Illumina RNA-Sequencing with competitive amplicon library preparation (n = 170) for samples C and D with correction for platform and assay specific bias.

    Journal: PLoS ONE

    Article Title: Targeted RNA-Sequencing with Competitive Multiplex-PCR Amplicon Libraries

    doi: 10.1371/journal.pone.0079120

    Figure Lengend Snippet: Cross-platform comparison of competitive amplicon library preparation with TaqMan qPCR and Illumina RNA-Sequencing. a ) Comparison of TaqMan qPCR with competitive amplicon library preparation (n = 146) for samples A and B without correction for systematic biases. Data is normalized to a median relative abundance. b ) Comparison of Illumina RNA-Sequencing with competitive amplicon library preparation (n = 170) for samples A and B without correction for systematic biases. Data is normalized to a median relative abundance. For a) and b), Spearman’s rank correlation coefficient is noted (r s ). The average of differences for measurements of samples A and B between competitive amplicon library preparation and TaqMan qPCR ( Figure S1 ) or Illumina RNA-sequencing ( Figure S2 ) was determined for each endogenous target; and to illustrate the systematic bias away from the regression line, data points for MMP2 have been highlighted in orange. This difference was subtracted from TaqMan qPCR or Illumina RNA-sequencing measurements for samples C and D and plotted (X-axis). Competitive amplicon library preparation measurements of C and D are plotted on the Y-axis. c) Comparison of TaqMan qPCR with competitive amplicon library preparation (n = 146) for samples C and D with correction for platform and assay specific bias. d) Comparison of Illumina RNA-Sequencing with competitive amplicon library preparation (n = 170) for samples C and D with correction for platform and assay specific bias.

    Article Snippet: We then evaluated the method for: a) accuracy and reproducibility of nucleic acid abundance measurement on different days within individual test sites, between test sites, and between different preparations of libraries, b) inter-platform concordance with TaqMan qPCR (MAQC-I study) and traditional RNA-sequencing library preparation using Illumina NGS kits (SEQC study), as well as c) reduced number of sequencing reads required for quantification.

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

    a Comparison of single-cell median and population-level RNA-Seq profiles for cells originating from the U87-exclusive lane in Experiment 1. Each data point was obtained by constructing a median profile from a given number of cells and repeating this 10 times by random sampling with replacement to obtain a median Pearson correlation coefficient and error bar (SEM). This exercise was repeated for comparison to both the U87 and MCF10a bulk RNA-Seq profiles to demonstrate better concordance between the U87 single-cell profiles and the U87 bulk profile. b Same as ( a ), but for single-cell profiles in the MCF10a-exlusive lane. c We conducted differential expression analysis to obtain cell type-specific gene sets for the U87 and MCF10a cells based on single-cell profiles from the pure-cell lanes. Here, we show a histogram of log-ratio of the coefficients of variation (CVs) for the cell type-specific gene sets between the mixed lane profiles and the profiles from the respective pure lanes. As expected, the heterogeneity given by CV is greater for cells in the mixed lanes than in the cell type-exclusive lanes for the cell type-specific genes

    Journal: Genome Biology

    Article Title: Scalable microfluidics for single-cell RNA printing and sequencing

    doi: 10.1186/s13059-015-0684-3

    Figure Lengend Snippet: a Comparison of single-cell median and population-level RNA-Seq profiles for cells originating from the U87-exclusive lane in Experiment 1. Each data point was obtained by constructing a median profile from a given number of cells and repeating this 10 times by random sampling with replacement to obtain a median Pearson correlation coefficient and error bar (SEM). This exercise was repeated for comparison to both the U87 and MCF10a bulk RNA-Seq profiles to demonstrate better concordance between the U87 single-cell profiles and the U87 bulk profile. b Same as ( a ), but for single-cell profiles in the MCF10a-exlusive lane. c We conducted differential expression analysis to obtain cell type-specific gene sets for the U87 and MCF10a cells based on single-cell profiles from the pure-cell lanes. Here, we show a histogram of log-ratio of the coefficients of variation (CVs) for the cell type-specific gene sets between the mixed lane profiles and the profiles from the respective pure lanes. As expected, the heterogeneity given by CV is greater for cells in the mixed lanes than in the cell type-exclusive lanes for the cell type-specific genes

    Article Snippet: We prepared a bulk RNA-Seq library from approximately 107 U87 cells using the TruSeq RNA-Seq library preparation kit (Illumina) and sequenced the library to a depth of approximately 30 M, 100-base single-end reads on an Illumina HiSeq 2500.

    Techniques: RNA Sequencing Assay, Sampling, Expressing

    Cell type separation by single-cell RNA-Seq. a iPAGE gene ontology/pathway analysis based on rank-ordering of differentially expressed genes using +/−(1-p) where p is the P value for differential expression between the U87- and MCF10a-exclusive lanes given by the Wilcoxin rank-sum test. Values are positive for genes more highly expressed in U87 and negative for genes more highly expressed in MCF10a. b t-SNE clustering of 396 single-cell profiles based on the differentially expressed genes color-coated by the lane-of-origin of each profile. Two clear spatial clusters form and each is predominantly associated with a specific cell type-exclusive lane. c The same t-SNE clustering shown in ( b ) but color-coated with a score indicating expression of the U87-specific genes vs. the MCF10a-specific genes. The score is based on the relative rank ordering of U87- and MCF10a-specific genes in each cell (see Methods )

    Journal: Genome Biology

    Article Title: Scalable microfluidics for single-cell RNA printing and sequencing

    doi: 10.1186/s13059-015-0684-3

    Figure Lengend Snippet: Cell type separation by single-cell RNA-Seq. a iPAGE gene ontology/pathway analysis based on rank-ordering of differentially expressed genes using +/−(1-p) where p is the P value for differential expression between the U87- and MCF10a-exclusive lanes given by the Wilcoxin rank-sum test. Values are positive for genes more highly expressed in U87 and negative for genes more highly expressed in MCF10a. b t-SNE clustering of 396 single-cell profiles based on the differentially expressed genes color-coated by the lane-of-origin of each profile. Two clear spatial clusters form and each is predominantly associated with a specific cell type-exclusive lane. c The same t-SNE clustering shown in ( b ) but color-coated with a score indicating expression of the U87-specific genes vs. the MCF10a-specific genes. The score is based on the relative rank ordering of U87- and MCF10a-specific genes in each cell (see Methods )

    Article Snippet: We prepared a bulk RNA-Seq library from approximately 107 U87 cells using the TruSeq RNA-Seq library preparation kit (Illumina) and sequenced the library to a depth of approximately 30 M, 100-base single-end reads on an Illumina HiSeq 2500.

    Techniques: RNA Sequencing Assay, Expressing

    Improved comparability across degradation effects and library preparation methods. ( a ) Correlation of unnormalized reads from normally processed iPSC RNA (HICL) and in-process degraded iPSC RNA (HICL_D). Reads for 51,552 loci over the read cutoff of 40 were plotted. ( b ) Correlation of SNV-normalized reads from the HICL and HICL_D samples. ( c ) Agreement of estimates for gene expression in HICL and HICL_D samples estimated by unnormalized reads and SNV-normalized reads. ( d ) Correlation of unnormalized reads generated by TruSeq method (HICL) and NexTera method (HICL_N). Reads for 53,585 loci were plotted. ( e ) Correlation of SNV-normalized reads from the HICL and HICL_N samples. ( f ) Agreement of estimates for gene expression in HICL and HICL_N samples estimated by unnormalized reads and SNV-normalized reads.

    Journal: Scientific Reports

    Article Title: Normalization of human RNA-seq experiments using chimpanzee RNA as a spike-in standard

    doi: 10.1038/srep31923

    Figure Lengend Snippet: Improved comparability across degradation effects and library preparation methods. ( a ) Correlation of unnormalized reads from normally processed iPSC RNA (HICL) and in-process degraded iPSC RNA (HICL_D). Reads for 51,552 loci over the read cutoff of 40 were plotted. ( b ) Correlation of SNV-normalized reads from the HICL and HICL_D samples. ( c ) Agreement of estimates for gene expression in HICL and HICL_D samples estimated by unnormalized reads and SNV-normalized reads. ( d ) Correlation of unnormalized reads generated by TruSeq method (HICL) and NexTera method (HICL_N). Reads for 53,585 loci were plotted. ( e ) Correlation of SNV-normalized reads from the HICL and HICL_N samples. ( f ) Agreement of estimates for gene expression in HICL and HICL_N samples estimated by unnormalized reads and SNV-normalized reads.

    Article Snippet: Preparation of NGS library Normal NGS library was constructed using TruSeq RNA Sample Preparation Kit v2 (Illumina) following the manufacturer’s instruction starting from 2 μg of total RNA.

    Techniques: Expressing, Generated

    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 (NEB) with 4 ng of RNA,NuGEN Ovation® RNA-Seq System V2 with 4 ng of RNA (NuGEN) with 4 ng of RNA, TaKaRa SMARTer® Stranded Total RNA-Seq Kit-Pico Input Mammalian with 4 ng of RNA (SMARTer), TaKaRa SMART-Seq® v4 Ultra® Low Input RNA Kit for Sequencing with 4 ng and 0.25 ng of RNA (SMARTseq4 and SMARTseq0.25) and Illumina TruSeq RNA Library Prep Kit v2 with 4 ng and 70 ng of RNA (TruSeq4 and TruSeq70).

    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 (NEB) with 4 ng of RNA,NuGEN Ovation® RNA-Seq System V2 with 4 ng of RNA (NuGEN) with 4 ng of RNA, TaKaRa SMARTer® Stranded Total RNA-Seq Kit-Pico Input Mammalian with 4 ng of RNA (SMARTer), TaKaRa SMART-Seq® v4 Ultra® Low Input RNA Kit for Sequencing with 4 ng and 0.25 ng of RNA (SMARTseq4 and SMARTseq0.25) and Illumina TruSeq RNA Library Prep Kit v2 with 4 ng and 70 ng of RNA (TruSeq4 and TruSeq70).

    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 (NEB) with 4 ng of RNA,NuGEN Ovation® RNA-Seq System V2 with 4 ng of RNA (NuGEN) with 4 ng of RNA, TaKaRa SMARTer® Stranded Total RNA-Seq Kit-Pico Input Mammalian with 4 ng of RNA (SMARTer), TaKaRa SMART-Seq® v4 Ultra® Low Input RNA Kit for Sequencing with 4 ng and 0.25 ng of RNA (SMARTseq4 and SMARTseq0.25) and Illumina TruSeq RNA Library Prep Kit v2 with 4 ng and 70 ng of RNA (TruSeq4 and TruSeq70).

    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 (NEB) with 4 ng of RNA,NuGEN Ovation® RNA-Seq System V2 with 4 ng of RNA (NuGEN) with 4 ng of RNA, TaKaRa SMARTer® Stranded Total RNA-Seq Kit-Pico Input Mammalian with 4 ng of RNA (SMARTer), TaKaRa SMART-Seq® v4 Ultra® Low Input RNA Kit for Sequencing with 4 ng and 0.25 ng of RNA (SMARTseq4 and SMARTseq0.25) and Illumina TruSeq RNA Library Prep Kit v2 with 4 ng and 70 ng of RNA (TruSeq4 and TruSeq70).

    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 (NEB) with 4 ng of RNA,NuGEN Ovation® RNA-Seq System V2 with 4 ng of RNA (NuGEN) with 4 ng of RNA, TaKaRa SMARTer® Stranded Total RNA-Seq Kit-Pico Input Mammalian with 4 ng of RNA (SMARTer), TaKaRa SMART-Seq® v4 Ultra® Low Input RNA Kit for Sequencing with 4 ng and 0.25 ng of RNA (SMARTseq4 and SMARTseq0.25) and Illumina TruSeq RNA Library Prep Kit v2 with 4 ng and 70 ng of RNA (TruSeq4 and TruSeq70).

    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 (NEB) with 4 ng of RNA,NuGEN Ovation® RNA-Seq System V2 with 4 ng of RNA (NuGEN) with 4 ng of RNA, TaKaRa SMARTer® Stranded Total RNA-Seq Kit-Pico Input Mammalian with 4 ng of RNA (SMARTer), TaKaRa SMART-Seq® v4 Ultra® Low Input RNA Kit for Sequencing with 4 ng and 0.25 ng of RNA (SMARTseq4 and SMARTseq0.25) and Illumina TruSeq RNA Library Prep Kit v2 with 4 ng and 70 ng of RNA (TruSeq4 and TruSeq70).

    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 (NEB) with 4 ng of RNA,NuGEN Ovation® RNA-Seq System V2 with 4 ng of RNA (NuGEN) with 4 ng of RNA, TaKaRa SMARTer® Stranded Total RNA-Seq Kit-Pico Input Mammalian with 4 ng of RNA (SMARTer), TaKaRa SMART-Seq® v4 Ultra® Low Input RNA Kit for Sequencing with 4 ng and 0.25 ng of RNA (SMARTseq4 and SMARTseq0.25) and Illumina TruSeq RNA Library Prep Kit v2 with 4 ng and 70 ng of RNA (TruSeq4 and TruSeq70).

    Techniques: