total rna  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier
Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93
    Name:
    NEBNext Ultra II RNA Library Prep with Sample Purification Beads
    Description:
    NEBNext Ultra II RNA Library Prep with Sample Purification Beads 96 rxns
    Catalog Number:
    e7775l
    Price:
    3350
    Size:
    96 rxns
    Category:
    mRNA Template Preparation for PCR
    Buy from Supplier


    Structured Review

    New England Biolabs total rna
    NEBNext Ultra II RNA Library Prep with Sample Purification Beads
    NEBNext Ultra II RNA Library Prep with Sample Purification Beads 96 rxns
    https://www.bioz.com/result/total rna/product/New England Biolabs
    Average 93 stars, based on 4206 article reviews
    Price from $9.99 to $1999.99
    total rna - by Bioz Stars, 2020-09
    93/100 stars

    Images

    1) Product Images from "Comparative Evaluation of cDNA Library Construction Approaches for RNA-Seq Analysis from Low RNA-Content Human Specimens"

    Article Title: Comparative Evaluation of cDNA Library Construction Approaches for RNA-Seq Analysis from Low RNA-Content Human Specimens

    Journal: Journal of microbiological methods

    doi: 10.1016/j.mimet.2018.10.008

    Overlap of the S. sanguinis expressed non-rRNA genes between the two library generation methods using the sonicate fluid sample (a) and the synovial fluid sample (b); and between the two sample types processed by the Illumina TruSeq Stranded Total RNA kit (c) and the NuGEN Ovation SoLo RNA-Seq System (d).
    Figure Legend Snippet: Overlap of the S. sanguinis expressed non-rRNA genes between the two library generation methods using the sonicate fluid sample (a) and the synovial fluid sample (b); and between the two sample types processed by the Illumina TruSeq Stranded Total RNA kit (c) and the NuGEN Ovation SoLo RNA-Seq System (d).

    Techniques Used: RNA Sequencing Assay

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky603

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

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

    3) Product Images from "Sensory neuron lineage mapping and manipulation in the Drosophila olfactory system"

    Article Title: Sensory neuron lineage mapping and manipulation in the Drosophila olfactory system

    Journal: Nature Communications

    doi: 10.1038/s41467-019-08345-4

    An OSN lineage-specific driver. a Top row: developmental expression of the nonimmortalized GMR82D08-GAL4 (hereafter, at1 driver) using a myr:GFP reporter (green) in the antennal disc SOPs (region marked by α-Dac (blue)) during late larval/early pupal stages. Bottom row: the at1 driver is expressed in the daughter cells of these SOPs in the developing pupal antenna but progressively loses its expression from 20 h APF as OSNs differentiate (visualized with the neuronal marker α-Elav (magenta)). Scale bar = 20 µm in this and other panels. b Immortalization of the at1 driver reveals labeling of clusters of cells in the adult antenna by an rCD2:GFP reporter (green). RNA fluorescence in situ hybridization demonstrates that a single cell within each cluster (arrowheads in the inset images) expresses Or67d mRNA (magenta). c Representative example of a single sensillum in the adult antenna labeled by the immortalized at1 driver, viewed at three focal planes. There is a single Or67d mRNA-positive OSN (cell 1, arrowhead), flanked by four non-neuronal support cells (cells 2–5). d
    Figure Legend Snippet: An OSN lineage-specific driver. a Top row: developmental expression of the nonimmortalized GMR82D08-GAL4 (hereafter, at1 driver) using a myr:GFP reporter (green) in the antennal disc SOPs (region marked by α-Dac (blue)) during late larval/early pupal stages. Bottom row: the at1 driver is expressed in the daughter cells of these SOPs in the developing pupal antenna but progressively loses its expression from 20 h APF as OSNs differentiate (visualized with the neuronal marker α-Elav (magenta)). Scale bar = 20 µm in this and other panels. b Immortalization of the at1 driver reveals labeling of clusters of cells in the adult antenna by an rCD2:GFP reporter (green). RNA fluorescence in situ hybridization demonstrates that a single cell within each cluster (arrowheads in the inset images) expresses Or67d mRNA (magenta). c Representative example of a single sensillum in the adult antenna labeled by the immortalized at1 driver, viewed at three focal planes. There is a single Or67d mRNA-positive OSN (cell 1, arrowhead), flanked by four non-neuronal support cells (cells 2–5). d

    Techniques Used: Expressing, Marker, Labeling, Fluorescence, In Situ Hybridization

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky156

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

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

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

    Techniques Used: Modification, Expressing, RNA Sequencing Assay

    5) Product Images from "Sensory Experience Remodels Genome Architecture in Neural Circuit to Drive Motor Learning"

    Article Title: Sensory Experience Remodels Genome Architecture in Neural Circuit to Drive Motor Learning

    Journal: Nature

    doi: 10.1038/s41586-019-1190-7

    Optostimulation of granule neurons potentiates CS pathway-regulated gene modules ( a ) UCSC genome browser tracks of chromatin-bound (Chrom-Seq) and nucleocytoplasmic (NucCyto-Seq) RNA at the fosl2 locus upon optostimulation of granule neurons in the ADCV in mice. The chromatin-bound fraction contained immature unspliced RNA and the nucleocytoplasmic fraction contained spliced mature RNA. ( b-d ) Comparisons of the log2 fold change in chromatin-bound RNA and the log2 fold change in nucleocytoplasmic RNA upon optostimulation of granule neurons together with the log2 fold change in total RNA upon sensorimotor stimulation in the ADCV in mice (n=4,4,18) mice for chromatin, nucleocytoplasmic, total RNA). Data show mean ± standard error. ( e ) Pulse chase analyses were performed by optogenetically stimulating granule neurons in the ADCV for 10 minutes and returning mice to their homecage for 10, 50, or 140 minutes. The ADCV of optostimulated or unstimulated control mice was then subjected to RNA-Seq using the chromatin-bound (nascent) or nucleocytoplasmic (mature) fractions. ( f-i ) Time course of chromatin-bound (nascent) or nucleocytoplasmic (mature) RNA expression following optostimulation of granule neurons as in (e) (n=2 mice).
    Figure Legend Snippet: Optostimulation of granule neurons potentiates CS pathway-regulated gene modules ( a ) UCSC genome browser tracks of chromatin-bound (Chrom-Seq) and nucleocytoplasmic (NucCyto-Seq) RNA at the fosl2 locus upon optostimulation of granule neurons in the ADCV in mice. The chromatin-bound fraction contained immature unspliced RNA and the nucleocytoplasmic fraction contained spliced mature RNA. ( b-d ) Comparisons of the log2 fold change in chromatin-bound RNA and the log2 fold change in nucleocytoplasmic RNA upon optostimulation of granule neurons together with the log2 fold change in total RNA upon sensorimotor stimulation in the ADCV in mice (n=4,4,18) mice for chromatin, nucleocytoplasmic, total RNA). Data show mean ± standard error. ( e ) Pulse chase analyses were performed by optogenetically stimulating granule neurons in the ADCV for 10 minutes and returning mice to their homecage for 10, 50, or 140 minutes. The ADCV of optostimulated or unstimulated control mice was then subjected to RNA-Seq using the chromatin-bound (nascent) or nucleocytoplasmic (mature) fractions. ( f-i ) Time course of chromatin-bound (nascent) or nucleocytoplasmic (mature) RNA expression following optostimulation of granule neurons as in (e) (n=2 mice).

    Techniques Used: Mouse Assay, Pulse Chase, RNA Sequencing Assay, RNA Expression

    6) Product Images from "A ligation-based single-stranded library preparation method to analyze cell-free DNA and synthetic oligos"

    Article Title: A ligation-based single-stranded library preparation method to analyze cell-free DNA and synthetic oligos

    Journal: BMC Genomics

    doi: 10.1186/s12864-019-6355-0

    Standard NGS metrics for merged reads from SRSLY and NEBNext Ultra II libraries from healthy human cfDNA extracts H-69 and H-81. Unless otherwise stated, all libraries for each method were combined by cfDNA extract prior to analysis and filtered for PCR duplicates and a quality score equal to or greater than q20. ( a ) Insert distribution plots for cfDNA extracts H-69 and H-81, respectively. ( b ) Fold coverage by base percent across the human genome ( hg19 ) for SRSLY and NEBNext by cfDNA extract. Combined libraries were subsampled to similar read depth prior to fold coverage calculations. Subsampled depth was set at 295 M reads, the limit of sequenced reads for SRSLY-H-81. ( c ) Preseq complexity estimate for SRSLY and NEBNext by cfDNA extract. Three libraries of equivalent sequencing depth per method were combined to estimate complexity, since more libraries were made via SRSLY than NEBNext. Files containing the PCR duplicate reads were used to facilitate complexity estimates ( d ) Normalized coverage as a function of GC content over 100 bp sliding scale across the human genome for SRSLY and NEBNext by cfDNA extract. Green histogram represents the human genome GC across the 100 bp sliding window. ( e ) Normalized, log-transformed base composition at each position of read termini starting 2 bp upstream and extending to 34 bp downstream of read start site for combined cfDNA extracts for SRSLY and NEBNext. All reads regardless of insert length considered
    Figure Legend Snippet: Standard NGS metrics for merged reads from SRSLY and NEBNext Ultra II libraries from healthy human cfDNA extracts H-69 and H-81. Unless otherwise stated, all libraries for each method were combined by cfDNA extract prior to analysis and filtered for PCR duplicates and a quality score equal to or greater than q20. ( a ) Insert distribution plots for cfDNA extracts H-69 and H-81, respectively. ( b ) Fold coverage by base percent across the human genome ( hg19 ) for SRSLY and NEBNext by cfDNA extract. Combined libraries were subsampled to similar read depth prior to fold coverage calculations. Subsampled depth was set at 295 M reads, the limit of sequenced reads for SRSLY-H-81. ( c ) Preseq complexity estimate for SRSLY and NEBNext by cfDNA extract. Three libraries of equivalent sequencing depth per method were combined to estimate complexity, since more libraries were made via SRSLY than NEBNext. Files containing the PCR duplicate reads were used to facilitate complexity estimates ( d ) Normalized coverage as a function of GC content over 100 bp sliding scale across the human genome for SRSLY and NEBNext by cfDNA extract. Green histogram represents the human genome GC across the 100 bp sliding window. ( e ) Normalized, log-transformed base composition at each position of read termini starting 2 bp upstream and extending to 34 bp downstream of read start site for combined cfDNA extracts for SRSLY and NEBNext. All reads regardless of insert length considered

    Techniques Used: Next-Generation Sequencing, Polymerase Chain Reaction, Sequencing, Transformation Assay

    cfDNA analysis. ( a ) Normalized genomic dinucleotide frequencies as a function of read length for SRSLY data for three discrete fragment lengths including 100 bp ± the read mapped coordinates. Read midpoint is centered at 0. Negative numbers denote genomic regions upstream (5-prime) of the midpoint and positive numbers denote genomic regions downstream (3-prime) of the midpoint. Input data is from the combined H-69 and H-81 SRSLY datasets. ( b ) Same as (a) except for NEBNext data. ( c ) Normalized genomic dinucleotide frequency as a function of read length for SRSLY data for the termini of three discrete fragment lengths including a 9 bp region into the read (positive numbers) and 10 bp outside the read (negative numbers). Read start and end coordinates are centered on 0. Input data is from the combined H-69 and H-81 SRSLY datasets. ( d ) Same as (c) except for NEBNext data. ( e ) Normalized WPS values (120 bp window; 120–180 bp fragments) for SRSLY data compared to sample CH01 [ 16 ] at the same pericentromeric locus on chromosome 12 used to initially showcase WPS. ( f ) Average normalized WPS score within ±1 kb of annotated CTCF binding sites for long fragment length binned data (120 bp window, 120–180 bp fragments) and short fragment length binned data (16 bp window, 35–80 bp fragments) for SRSLY data compared to sample CH01 [ 16 ]
    Figure Legend Snippet: cfDNA analysis. ( a ) Normalized genomic dinucleotide frequencies as a function of read length for SRSLY data for three discrete fragment lengths including 100 bp ± the read mapped coordinates. Read midpoint is centered at 0. Negative numbers denote genomic regions upstream (5-prime) of the midpoint and positive numbers denote genomic regions downstream (3-prime) of the midpoint. Input data is from the combined H-69 and H-81 SRSLY datasets. ( b ) Same as (a) except for NEBNext data. ( c ) Normalized genomic dinucleotide frequency as a function of read length for SRSLY data for the termini of three discrete fragment lengths including a 9 bp region into the read (positive numbers) and 10 bp outside the read (negative numbers). Read start and end coordinates are centered on 0. Input data is from the combined H-69 and H-81 SRSLY datasets. ( d ) Same as (c) except for NEBNext data. ( e ) Normalized WPS values (120 bp window; 120–180 bp fragments) for SRSLY data compared to sample CH01 [ 16 ] at the same pericentromeric locus on chromosome 12 used to initially showcase WPS. ( f ) Average normalized WPS score within ±1 kb of annotated CTCF binding sites for long fragment length binned data (120 bp window, 120–180 bp fragments) and short fragment length binned data (16 bp window, 35–80 bp fragments) for SRSLY data compared to sample CH01 [ 16 ]

    Techniques Used: Binding Assay

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

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

    Journal: Cancer Cell

    doi: 10.1016/j.ccell.2018.08.017

    Enhanced Short- and Long-Term Reactivity of CAR19-iNKT Cells against B Lineage Malignancies (A) Second- and third-generation CAR19-T and CAR19-iNKT cell expansion (fold change) and absolute cell numbers (cell count) over a period of 3 weeks (n = 4). p value is for CAR19-iNKT versus CAR19-T cells using Friedman test. Error bars represent SEM. (B) Proliferation analysis of second- and third-generation CAR19-T and CAR19-iNKT cells in the presence (stimulated) or not (resting) of irradiated CD1d + CD19 + (C1R-CD1d) cells over 7 days. p value is for CAR19-iNKT versus CAR19-T cells using Friedman test. (C) Cytotoxicity of third-generation CAR19-T and -NKT cells against C1R-CD1d (representative of n = 3) and Farage lymphoma cell lines (representative of n = 2) pre-loaded or not with αGalCer. Error bars represent SEM of triplicate assays. (D) IncuCyte images of representative wells showing the final effector (gray) and live target cells (red) after 7 days. Effectors were second-generation CAR19-T and CAR19-NKT cells. Targets were CD19 + ARH-77-CD1d cells expressing mCherry red fluorescent protein. Scale bar represents 400 μm. (E) Seven-day trajectory of effector and target cell proliferation and elimination respectively as per (D). p value is for CAR19-iNKT versus CAR19-T cells using Friedman test. (F) Cytotoxicity of second-generation CAR19-iNKT, CAR19-T, and of untransduced iNKT cells against lymphoma cells from one patient with mantle cell lymphoma (MCL; top) and two patients with marginal zone B lymphoma (MZL; bottom) using three different T/iNKT cell healthy donors (A, B, and C). Error bars represent SEM of triplicate assays. ∗∗ p
    Figure Legend Snippet: Enhanced Short- and Long-Term Reactivity of CAR19-iNKT Cells against B Lineage Malignancies (A) Second- and third-generation CAR19-T and CAR19-iNKT cell expansion (fold change) and absolute cell numbers (cell count) over a period of 3 weeks (n = 4). p value is for CAR19-iNKT versus CAR19-T cells using Friedman test. Error bars represent SEM. (B) Proliferation analysis of second- and third-generation CAR19-T and CAR19-iNKT cells in the presence (stimulated) or not (resting) of irradiated CD1d + CD19 + (C1R-CD1d) cells over 7 days. p value is for CAR19-iNKT versus CAR19-T cells using Friedman test. (C) Cytotoxicity of third-generation CAR19-T and -NKT cells against C1R-CD1d (representative of n = 3) and Farage lymphoma cell lines (representative of n = 2) pre-loaded or not with αGalCer. Error bars represent SEM of triplicate assays. (D) IncuCyte images of representative wells showing the final effector (gray) and live target cells (red) after 7 days. Effectors were second-generation CAR19-T and CAR19-NKT cells. Targets were CD19 + ARH-77-CD1d cells expressing mCherry red fluorescent protein. Scale bar represents 400 μm. (E) Seven-day trajectory of effector and target cell proliferation and elimination respectively as per (D). p value is for CAR19-iNKT versus CAR19-T cells using Friedman test. (F) Cytotoxicity of second-generation CAR19-iNKT, CAR19-T, and of untransduced iNKT cells against lymphoma cells from one patient with mantle cell lymphoma (MCL; top) and two patients with marginal zone B lymphoma (MZL; bottom) using three different T/iNKT cell healthy donors (A, B, and C). Error bars represent SEM of triplicate assays. ∗∗ p

    Techniques Used: Cell Counting, Irradiation, Expressing

    8) Product Images from "In vivo CRISPR screening in CD8 T cells with AAV-Sleeping Beauty hybrid vectors identifies membrane targets for improving immunotherapy for glioblastoma"

    Article Title: In vivo CRISPR screening in CD8 T cells with AAV-Sleeping Beauty hybrid vectors identifies membrane targets for improving immunotherapy for glioblastoma

    Journal: Nature biotechnology

    doi: 10.1038/s41587-019-0246-4

    Single-cell RNA-seq and bulk mRNA-seq analysis of Pdia3 knockout in CD8 + T cells ( a ) t-SNE plot of sample distribution based on the transcriptome of 9,193 single cells from AAV-sgPdia3 and AAV-Vector treated CD8 + T cells. ( b ) Bubble-rank plot of differential gene expression of scRNA-seq. Delta-mean is the difference of mean expression value between AAV-sgPdia3 and AAV-Vector treated single CD8 + T cells (n = 3 each group). Differential expression: Two-sided Wilcoxon signed-rank test by gene, with p-values adjusted by Benjamini Hochberg. Statistical significance is scaled by –log10, p -value as shown in the size key. ( c ) A volcano plot of all differentially expressed genes between AAV-Vector and AAV-sgPdia3 transduced mouse primary CD8 + T cells (n = 3 biological replicates). Differential gene expression was performed with Sleuth using Wald test, the FDR adjusted q-value was used for the plot. ( d ) Heatmap of representative immune-related differentially expressed genes between AAV-Vector and AAV-sgPdia3 transduced mouse primary CD8 + T cells (n = 3 biological replicates). ( e ) RT-qPCR validation of the scRNA-seq and bulk mRNA-seq results confirmed the upregulation of granzyme genes upon AAV-sgPdia3 perturbation (n = 4). Unpaired t test, two-tailed. * p
    Figure Legend Snippet: Single-cell RNA-seq and bulk mRNA-seq analysis of Pdia3 knockout in CD8 + T cells ( a ) t-SNE plot of sample distribution based on the transcriptome of 9,193 single cells from AAV-sgPdia3 and AAV-Vector treated CD8 + T cells. ( b ) Bubble-rank plot of differential gene expression of scRNA-seq. Delta-mean is the difference of mean expression value between AAV-sgPdia3 and AAV-Vector treated single CD8 + T cells (n = 3 each group). Differential expression: Two-sided Wilcoxon signed-rank test by gene, with p-values adjusted by Benjamini Hochberg. Statistical significance is scaled by –log10, p -value as shown in the size key. ( c ) A volcano plot of all differentially expressed genes between AAV-Vector and AAV-sgPdia3 transduced mouse primary CD8 + T cells (n = 3 biological replicates). Differential gene expression was performed with Sleuth using Wald test, the FDR adjusted q-value was used for the plot. ( d ) Heatmap of representative immune-related differentially expressed genes between AAV-Vector and AAV-sgPdia3 transduced mouse primary CD8 + T cells (n = 3 biological replicates). ( e ) RT-qPCR validation of the scRNA-seq and bulk mRNA-seq results confirmed the upregulation of granzyme genes upon AAV-sgPdia3 perturbation (n = 4). Unpaired t test, two-tailed. * p

    Techniques Used: RNA Sequencing Assay, Knock-Out, Plasmid Preparation, Expressing, Quantitative RT-PCR, Two Tailed Test

    9) Product Images from "Characterization of human mosaic Rett syndrome brain tissue by single-nucleus RNA sequencing"

    Article Title: Characterization of human mosaic Rett syndrome brain tissue by single-nucleus RNA sequencing

    Journal: Nature neuroscience

    doi: 10.1038/s41593-018-0270-6

    Cell-type-specific DNA methylation patterns predict gene misregulation in Rett syndrome. For each graph in A-B and D-E, mean fold-change in gene expression of R255X MECP2 nuclei compared to WT nuclei (R255X v WT) or of two groups of the respective cell type that were randomly assigned transcriptotypes (Random) is binned according to the fraction of gene body DNA methylation (mCH/CH). Gene expression changes (FDR
    Figure Legend Snippet: Cell-type-specific DNA methylation patterns predict gene misregulation in Rett syndrome. For each graph in A-B and D-E, mean fold-change in gene expression of R255X MECP2 nuclei compared to WT nuclei (R255X v WT) or of two groups of the respective cell type that were randomly assigned transcriptotypes (Random) is binned according to the fraction of gene body DNA methylation (mCH/CH). Gene expression changes (FDR

    Techniques Used: DNA Methylation Assay, Expressing

    10) Product Images from "RiboTag translatomic profiling of Drosophila oenocytes under aging and induced oxidative stress"

    Article Title: RiboTag translatomic profiling of Drosophila oenocytes under aging and induced oxidative stress

    Journal: BMC Genomics

    doi: 10.1186/s12864-018-5404-4

    Differential gene expression analysis reveals common and distinct translational regulation by aging and oxidative stress. a Principal component analysis (PCA) on four oenocyte translatomes. b-d Correlation analysis on the gene expression between H2O-Young and H2O-Aged; H2O-Young and PQ-Young; H2O-Aged and PQ-Aged. Log 10 (FPKM) was used in the analysis e-f Venn diagram and GO terms for the genes commonly and differentially regulated by aging and paraquat. g-h Venn diagram and GO terms for the genes commonly and differentially regulated by paraquat at young and old ages. i Hierarchy clustering analysis on oenocyte translatome. j Gene ontology analysis on cluster 3, 5, 10 in panel ( i ). OXPHOS: oxidative phosphorylation. FA: fatty acid
    Figure Legend Snippet: Differential gene expression analysis reveals common and distinct translational regulation by aging and oxidative stress. a Principal component analysis (PCA) on four oenocyte translatomes. b-d Correlation analysis on the gene expression between H2O-Young and H2O-Aged; H2O-Young and PQ-Young; H2O-Aged and PQ-Aged. Log 10 (FPKM) was used in the analysis e-f Venn diagram and GO terms for the genes commonly and differentially regulated by aging and paraquat. g-h Venn diagram and GO terms for the genes commonly and differentially regulated by paraquat at young and old ages. i Hierarchy clustering analysis on oenocyte translatome. j Gene ontology analysis on cluster 3, 5, 10 in panel ( i ). OXPHOS: oxidative phosphorylation. FA: fatty acid

    Techniques Used: Expressing

    11) Product Images from "MicroRNAs Circulate in the Hemolymph of Drosophila and Accumulate Relative to Tissue microRNAs in an Age-Dependent Manner"

    Article Title: MicroRNAs Circulate in the Hemolymph of Drosophila and Accumulate Relative to Tissue microRNAs in an Age-Dependent Manner

    Journal: Genomics Insights

    doi: 10.4137/GEI.S38147

    Presence of stable miRNAs in Drosophila melanogaster hemolymph. ( A , B ) Clear HL droplets extruded from fly head and thorax. ( C – F ) Real-time qPCR amplification of selected HL miRNAs and mRNAs. Total RNA including small RNA was extracted from HL samples and analyzed with qPCR to measure the levels of miRNAs and mRNAs. The y -axis represents the relative fluorescence units (RFU) in a semi-log scale. The x -axis represents the cycle at which fluorescence was detected above an automatically determined threshold. ( C ) Amplification plots for miR-14, miR-8, and miR-184 measured in a representative HL sample. ( D ) Amplification plots for miR-14, let-7, bantam, and spiked-in synthetic C. elegans cel-miR-39 RNA in representative HL sample. ( E ) The amplification plots for tubulin, actin, and gapdh mRNAs determined by qPCR using total RNA from HL and S2 cells. Sample from S2 cells are used as a positive control for detecting Drosophila mRNAs by qPCR. The amplification curves of all three mRNAs are superimposed on one another, reflecting the presence of similar amounts of these mRNA in S2 cells. Amplification curves from HL samples show that fluorescent products appear after about 30 cycles, reflecting the significantly lower abundance of these mRNAs in HL relative to S2 cells. ( F ) The cycle threshold (Ct) fold-change of selected miRNA amplified in the absence or presence of RNase A and DNase I. Total RNA was extracted from HL samples spiked with 10 fmoles of cel-miR-39 RNA. The x -axis represents the ratio of raw Ct values from control samples divided by raw Ct values from samples incubated with RNase A and DNase I. The significantly higher magnitude of the Ct fold change of the spiked-in synthetic miRNA relative to those of the miRNA indicates that the HL-miRNA are present in nuclease-resistant, stable form.
    Figure Legend Snippet: Presence of stable miRNAs in Drosophila melanogaster hemolymph. ( A , B ) Clear HL droplets extruded from fly head and thorax. ( C – F ) Real-time qPCR amplification of selected HL miRNAs and mRNAs. Total RNA including small RNA was extracted from HL samples and analyzed with qPCR to measure the levels of miRNAs and mRNAs. The y -axis represents the relative fluorescence units (RFU) in a semi-log scale. The x -axis represents the cycle at which fluorescence was detected above an automatically determined threshold. ( C ) Amplification plots for miR-14, miR-8, and miR-184 measured in a representative HL sample. ( D ) Amplification plots for miR-14, let-7, bantam, and spiked-in synthetic C. elegans cel-miR-39 RNA in representative HL sample. ( E ) The amplification plots for tubulin, actin, and gapdh mRNAs determined by qPCR using total RNA from HL and S2 cells. Sample from S2 cells are used as a positive control for detecting Drosophila mRNAs by qPCR. The amplification curves of all three mRNAs are superimposed on one another, reflecting the presence of similar amounts of these mRNA in S2 cells. Amplification curves from HL samples show that fluorescent products appear after about 30 cycles, reflecting the significantly lower abundance of these mRNAs in HL relative to S2 cells. ( F ) The cycle threshold (Ct) fold-change of selected miRNA amplified in the absence or presence of RNase A and DNase I. Total RNA was extracted from HL samples spiked with 10 fmoles of cel-miR-39 RNA. The x -axis represents the ratio of raw Ct values from control samples divided by raw Ct values from samples incubated with RNase A and DNase I. The significantly higher magnitude of the Ct fold change of the spiked-in synthetic miRNA relative to those of the miRNA indicates that the HL-miRNA are present in nuclease-resistant, stable form.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Fluorescence, Positive Control, Incubation

    Outline of the experimental design and workflow for the integrated analysis of the miRNA-Seq and mRNA-Seq data. Genes that are potentially regulated by the HL-miRNAs were determined by integrating miRNA-Seq and mRNA-Seq data using the following three steps: Step 1 (depicted in green): After identifying the HL-miRNAs enriched relative to BT in either young or old, or both young and old ( Tables 1 and 2 ), we retrieved their computationally predicted target genes from the m 3 RNA database ( http://m3rna.cnb.csic.es ). Step 2 (depicted in blue): mRNA-Seq was utilized to identify three groups of genes that were differentially expressed with age, either upregulated, downregulated, or unchanged in BT. Step 3 (depicted in red): The three groups of genes predicted in step1 as targets of HL-miRNAs were intersected with the three groups of genes identified in step 2. In the figure, ( A ) depicts the derivation of genes that are predicted to be targets of HL-miRNAs enriched in young which are also upregulated with age in BT. These genes (Supplementary Table 1 ) are likely upregulated in BT of old flies because their expression is no longer repressed by HL-miRNAs that are enriched only in young flies. ( B ) Depicts the derivation of genes which are the predicted to be targets of HL-miRNAs enriched in old which are also downregulated with age in BT. These genes (Supplementary Table 2 ) are likely downregulated in BT of old flies because their expression is repressed by HL-miRNAs which are enriched only in old flies. ( C ) Depicts the derivation of genes which are predicted to be targets of HL-miRNAs enriched in both young and old which also do not change expression with age in BT. These genes (Supplementary Table 3) do not change expression with age because the concentration of their regulatory miRNAs in HL does not change with age.
    Figure Legend Snippet: Outline of the experimental design and workflow for the integrated analysis of the miRNA-Seq and mRNA-Seq data. Genes that are potentially regulated by the HL-miRNAs were determined by integrating miRNA-Seq and mRNA-Seq data using the following three steps: Step 1 (depicted in green): After identifying the HL-miRNAs enriched relative to BT in either young or old, or both young and old ( Tables 1 and 2 ), we retrieved their computationally predicted target genes from the m 3 RNA database ( http://m3rna.cnb.csic.es ). Step 2 (depicted in blue): mRNA-Seq was utilized to identify three groups of genes that were differentially expressed with age, either upregulated, downregulated, or unchanged in BT. Step 3 (depicted in red): The three groups of genes predicted in step1 as targets of HL-miRNAs were intersected with the three groups of genes identified in step 2. In the figure, ( A ) depicts the derivation of genes that are predicted to be targets of HL-miRNAs enriched in young which are also upregulated with age in BT. These genes (Supplementary Table 1 ) are likely upregulated in BT of old flies because their expression is no longer repressed by HL-miRNAs that are enriched only in young flies. ( B ) Depicts the derivation of genes which are the predicted to be targets of HL-miRNAs enriched in old which are also downregulated with age in BT. These genes (Supplementary Table 2 ) are likely downregulated in BT of old flies because their expression is repressed by HL-miRNAs which are enriched only in old flies. ( C ) Depicts the derivation of genes which are predicted to be targets of HL-miRNAs enriched in both young and old which also do not change expression with age in BT. These genes (Supplementary Table 3) do not change expression with age because the concentration of their regulatory miRNAs in HL does not change with age.

    Techniques Used: Expressing, Concentration Assay

    12) Product Images from "Comparative RNA-Seq Analysis Reveals That Regulatory Network of Maize Root Development Controls the Expression of Genes in Response to N Stress"

    Article Title: Comparative RNA-Seq Analysis Reveals That Regulatory Network of Maize Root Development Controls the Expression of Genes in Response to N Stress

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0151697

    RNA-Seq analysis of rtcs and wild-type roots transcriptome under normal-N condition. (A) Venn diagram analysis of DEGs between wild-type and rtcs at four developmental time points. (B) Co-modulated differential expression transcription factors (TFs) between wild-type and rtcs at four time points. TFs were grouped by family and the number of differential expression TFs is indicated. Each column represents average expression differences across the TF family at one time point. (C) Clustering co-modulated DEGs based on the expression profiles (FPKM values were log10-transformed). The top GO terms and corresponding DEGs number are shown on the right side, “*” represents significant enrichment (FDR
    Figure Legend Snippet: RNA-Seq analysis of rtcs and wild-type roots transcriptome under normal-N condition. (A) Venn diagram analysis of DEGs between wild-type and rtcs at four developmental time points. (B) Co-modulated differential expression transcription factors (TFs) between wild-type and rtcs at four time points. TFs were grouped by family and the number of differential expression TFs is indicated. Each column represents average expression differences across the TF family at one time point. (C) Clustering co-modulated DEGs based on the expression profiles (FPKM values were log10-transformed). The top GO terms and corresponding DEGs number are shown on the right side, “*” represents significant enrichment (FDR

    Techniques Used: RNA Sequencing Assay, Expressing, Transformation Assay

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

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

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.00682

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

    Techniques Used: Isolation, Fluorescence, FACS, Purification

    14) Product Images from "Functional integration of “undead” neurons in the olfactory system"

    Article Title: Functional integration of “undead” neurons in the olfactory system

    Journal: Science Advances

    doi: 10.1126/sciadv.aaz7238

    Undead neurons express a subset of olfactory receptor genes. ( A ) Gene expression differences measured by RNA-seq (see Materials and Methods) between control and PCD-blocked antennae. The volcano plot shows the differential expression (on the x axis) of D. melanogaster Or , Ir , and Gr gene transcripts (each gene represented by a dot), as well as the four proapoptotic genes ( grim , rpr , hid , and skl ; red labels), plotted against the statistical significance (on the y axis). The mean expression level of individual genes across all samples is shown by the shading of the dot, as indicated by the gray scale on the right [units: log 2 (counts per million)]. Only chemosensory genes showing a > 1.5-fold increase in PCD-blocked antennae are labeled: Blue labels indicate genes whose expression in the antenna has previously been demonstrated by RNA in situ hybridization; magenta and green labels indicate receptors normally only expressed in the adult maxillary palps and larval dorsal organ, respectively; and black labels indicate receptors that are expressed in gustatory organs. The horizontal dashed line indicates a false discovery rate threshold of 5%. Data for all Or , Ir , Gr , and proapoptotic genes are provided in table S2. ( B ) Representative images of RNA FISH for the indicated Or genes in whole-mount antennae of control ( peb-Gal4/+ ) and PCD-blocked ( peb-Gal4/+;UAS-p35/+ ) animals. Scale bar, 10 μm. Quantifications of neuron numbers are shown at the bottom. *** indicates Or19a P = 6.526 × 10 −5 ( t test) [ n = 17 and 10 (control and PCD-blocked, respectively)], Or43a P = 5.888 × 10 −7 ( t test) ( n = 10 and 10), Or47a P = 3.088 × 10 −10 ( t test) ( n = 13 and 13), Or65b P = 2.2 × 10 −16 ( t test) ( n = 17 and 11) (see fig. S2A for additional examples). The pink dashed lines encircle cells in PCD-blocked antennae that express the visualized Or s outside their usual spatial domain (see also figs. S2B and S3A). Comparison of the variation in OSN number between control and PCD-blocked antennae indicated only one neuron type—of those in this panel and in figs. S2A and S4—displayed greater variance in PCD-blocked antennae (Or19a P = 0.01; F test for equality of variance, with Bonferroni correction for multiple comparisons). ( C ) Representative images of RNA FISH for the indicated Or genes in whole-mount antennae of control ( peb-Gal4/+ ) and PCD-blocked ( peb-Gal4/+;UAS-p35/+ ) animals. Scale bar, 10 μm. Quantifications of neuron numbers are shown at the bottom. *** indicates Or33a P = 1.812 × 10 −7 ( t test) ( n = 23 and 23), ns indicates Or85e P = 0.053 (Wilcoxon rank sum test) ( n = 12 and 22). We never detected any Or85e mRNA-positive neurons in control antennae but frequently detected one (or more) labeled cells in PCD-blocked antennae. ( D ) Schematic summarizing the normal olfactory organ/sensillum expression pattern of the subset of Or genes with higher expression in PCD-blocked antennae that display coexpression in wild-type neurons (highlighted in red; receptor genes showing no changes in transcript levels are in gray). The neuronal precursor identity of these OSNs is shown below. We represent Or69aA and Or69aB as distinct receptors here because, although they share 3′ exons, they are transcribed from different promoters and encode receptors with different odor specificities ( 15 , 56 ). (These isoforms were not, however, distinguished in the RNA-seq and RNA FISH analyses). ( E ) Representative images of combined RNA FISH for Or65a (green) and Or65b (magenta) in whole-mount antennae of control ( peb-Gal4/+ ) and PCD-blocked ( peb-Gal4/+;UAS-p35/+ ) animals ( n = 4 and 5, respectively), showing coexpression of these receptors in both endogenous and undead neurons. Scale bar, 10 μm. ( F ) Representative images of RNA FISH for Or85f and anti-GFP in whole-mount antennae of control ( peb-Gal4/+;Or49a-GFP/+ ) and PCD-blocked ( peb-Gal4/+;Or49a-GFP/UAS-p35 ) animals. Scale bar, 10 μm. Quantifications of coexpression are shown to the right. *** indicates Or49a- GFP + /Or85f mRNA − population P = 5.48 × 10 −12 ( t test) ( n = 16 and 15; control and PCD-blocked, respectively). The pink dashed line encircles cells in the PCD-blocked antenna that express Or49a -GFP outside the usual spatial domain (see also fig. S2B). We used an Or49a- GFP reporter due to our inability to reliably detect Or49a transcripts in situ; the higher number of Or49a- GFP + /Or85f mRNA − negative cells is not an artifact of the detection method, as an Or85f -GFP reporter revealed a similarly limited increase in neuron number (fig. S3B). ( G ) Representative images of combined RNA FISH for Or47a (green) and Or33b (magenta) in whole-mount antennae of control ( peb-Gal4/+ ) and PCD-blocked ( peb-Gal4/+;UAS-p35/+ ) animals. In control animals, Or33b is coexpressed with Or47a in the larval dorsal organ and is never detected in the antenna. In PCD-blocked antennae, Or33b - and Or47a -expressing undead neurons are almost completely nonoverlapping: 4% of Or33b -positive undead OSNs weakly coexpress Or47a ( n = 79 cells from 10 antennae). Scale bar, 10 μm.
    Figure Legend Snippet: Undead neurons express a subset of olfactory receptor genes. ( A ) Gene expression differences measured by RNA-seq (see Materials and Methods) between control and PCD-blocked antennae. The volcano plot shows the differential expression (on the x axis) of D. melanogaster Or , Ir , and Gr gene transcripts (each gene represented by a dot), as well as the four proapoptotic genes ( grim , rpr , hid , and skl ; red labels), plotted against the statistical significance (on the y axis). The mean expression level of individual genes across all samples is shown by the shading of the dot, as indicated by the gray scale on the right [units: log 2 (counts per million)]. Only chemosensory genes showing a > 1.5-fold increase in PCD-blocked antennae are labeled: Blue labels indicate genes whose expression in the antenna has previously been demonstrated by RNA in situ hybridization; magenta and green labels indicate receptors normally only expressed in the adult maxillary palps and larval dorsal organ, respectively; and black labels indicate receptors that are expressed in gustatory organs. The horizontal dashed line indicates a false discovery rate threshold of 5%. Data for all Or , Ir , Gr , and proapoptotic genes are provided in table S2. ( B ) Representative images of RNA FISH for the indicated Or genes in whole-mount antennae of control ( peb-Gal4/+ ) and PCD-blocked ( peb-Gal4/+;UAS-p35/+ ) animals. Scale bar, 10 μm. Quantifications of neuron numbers are shown at the bottom. *** indicates Or19a P = 6.526 × 10 −5 ( t test) [ n = 17 and 10 (control and PCD-blocked, respectively)], Or43a P = 5.888 × 10 −7 ( t test) ( n = 10 and 10), Or47a P = 3.088 × 10 −10 ( t test) ( n = 13 and 13), Or65b P = 2.2 × 10 −16 ( t test) ( n = 17 and 11) (see fig. S2A for additional examples). The pink dashed lines encircle cells in PCD-blocked antennae that express the visualized Or s outside their usual spatial domain (see also figs. S2B and S3A). Comparison of the variation in OSN number between control and PCD-blocked antennae indicated only one neuron type—of those in this panel and in figs. S2A and S4—displayed greater variance in PCD-blocked antennae (Or19a P = 0.01; F test for equality of variance, with Bonferroni correction for multiple comparisons). ( C ) Representative images of RNA FISH for the indicated Or genes in whole-mount antennae of control ( peb-Gal4/+ ) and PCD-blocked ( peb-Gal4/+;UAS-p35/+ ) animals. Scale bar, 10 μm. Quantifications of neuron numbers are shown at the bottom. *** indicates Or33a P = 1.812 × 10 −7 ( t test) ( n = 23 and 23), ns indicates Or85e P = 0.053 (Wilcoxon rank sum test) ( n = 12 and 22). We never detected any Or85e mRNA-positive neurons in control antennae but frequently detected one (or more) labeled cells in PCD-blocked antennae. ( D ) Schematic summarizing the normal olfactory organ/sensillum expression pattern of the subset of Or genes with higher expression in PCD-blocked antennae that display coexpression in wild-type neurons (highlighted in red; receptor genes showing no changes in transcript levels are in gray). The neuronal precursor identity of these OSNs is shown below. We represent Or69aA and Or69aB as distinct receptors here because, although they share 3′ exons, they are transcribed from different promoters and encode receptors with different odor specificities ( 15 , 56 ). (These isoforms were not, however, distinguished in the RNA-seq and RNA FISH analyses). ( E ) Representative images of combined RNA FISH for Or65a (green) and Or65b (magenta) in whole-mount antennae of control ( peb-Gal4/+ ) and PCD-blocked ( peb-Gal4/+;UAS-p35/+ ) animals ( n = 4 and 5, respectively), showing coexpression of these receptors in both endogenous and undead neurons. Scale bar, 10 μm. ( F ) Representative images of RNA FISH for Or85f and anti-GFP in whole-mount antennae of control ( peb-Gal4/+;Or49a-GFP/+ ) and PCD-blocked ( peb-Gal4/+;Or49a-GFP/UAS-p35 ) animals. Scale bar, 10 μm. Quantifications of coexpression are shown to the right. *** indicates Or49a- GFP + /Or85f mRNA − population P = 5.48 × 10 −12 ( t test) ( n = 16 and 15; control and PCD-blocked, respectively). The pink dashed line encircles cells in the PCD-blocked antenna that express Or49a -GFP outside the usual spatial domain (see also fig. S2B). We used an Or49a- GFP reporter due to our inability to reliably detect Or49a transcripts in situ; the higher number of Or49a- GFP + /Or85f mRNA − negative cells is not an artifact of the detection method, as an Or85f -GFP reporter revealed a similarly limited increase in neuron number (fig. S3B). ( G ) Representative images of combined RNA FISH for Or47a (green) and Or33b (magenta) in whole-mount antennae of control ( peb-Gal4/+ ) and PCD-blocked ( peb-Gal4/+;UAS-p35/+ ) animals. In control animals, Or33b is coexpressed with Or47a in the larval dorsal organ and is never detected in the antenna. In PCD-blocked antennae, Or33b - and Or47a -expressing undead neurons are almost completely nonoverlapping: 4% of Or33b -positive undead OSNs weakly coexpress Or47a ( n = 79 cells from 10 antennae). Scale bar, 10 μm.

    Techniques Used: Expressing, RNA Sequencing Assay, Labeling, RNA In Situ Hybridization, Fluorescence In Situ Hybridization, In Situ

    15) Product Images from "Selective release of circRNAs in platelet-derived extracellular vesicles"

    Article Title: Selective release of circRNAs in platelet-derived extracellular vesicles

    Journal: Journal of Extracellular Vesicles

    doi: 10.1080/20013078.2018.1424473

    Isolation of platelet-derived EVs. (a) Flow chart of the purification of platelet-derived microvesicles and exosomes, based on differential centrifugation (for details, see Materials and methods). (b) Electron microscopy of purified vesicles. Purified exosomes and microvesicles were counterstained by uranyl acetate. In addition, exosomes were stained with 10 nm gold-conjugated anti-CD63 antibody (in two different magnifications). Scale bar, 100 nm. (c) Characterisation of exosome preparation by Western blot. Exosomes were purified by sucrose-density centrifugation, and each fraction (#1–12, from top to bottom) was subjected to Western blot analysis, using the exosomal marker CD63, with calnexin as a negative control. Platelet extract was loaded as input control. Sucrose densities (in g/mL) of the individual fractions are indicated at the bottom. (d) CircRNAs cofractionate with exosomes. Total RNA was isolated from each fraction of the sucrose gradient (#1–12, from top to bottom), followed by RT-PCR, using circRNA-specific primers for GSE1 and NRIP1. M , markers (200, 100 bp). (e) Purified microvesicles and exosomes were analysed by dynamic light scattering, depicting the calculated size distribution.
    Figure Legend Snippet: Isolation of platelet-derived EVs. (a) Flow chart of the purification of platelet-derived microvesicles and exosomes, based on differential centrifugation (for details, see Materials and methods). (b) Electron microscopy of purified vesicles. Purified exosomes and microvesicles were counterstained by uranyl acetate. In addition, exosomes were stained with 10 nm gold-conjugated anti-CD63 antibody (in two different magnifications). Scale bar, 100 nm. (c) Characterisation of exosome preparation by Western blot. Exosomes were purified by sucrose-density centrifugation, and each fraction (#1–12, from top to bottom) was subjected to Western blot analysis, using the exosomal marker CD63, with calnexin as a negative control. Platelet extract was loaded as input control. Sucrose densities (in g/mL) of the individual fractions are indicated at the bottom. (d) CircRNAs cofractionate with exosomes. Total RNA was isolated from each fraction of the sucrose gradient (#1–12, from top to bottom), followed by RT-PCR, using circRNA-specific primers for GSE1 and NRIP1. M , markers (200, 100 bp). (e) Purified microvesicles and exosomes were analysed by dynamic light scattering, depicting the calculated size distribution.

    Techniques Used: Isolation, Derivative Assay, Purification, Centrifugation, Electron Microscopy, Staining, Western Blot, Marker, Negative Control, Reverse Transcription Polymerase Chain Reaction

    CircRNA-protein complexes in human platelets. Sedimentation profiles of circRNPs. Platelet extract and corresponding free RNA (prepared from the same extract) were fractionated by glycerol gradient centrifugation (fractions #1–12 from top to bottom), followed by RT-PCR analysis across the gradient for five abundant circRNAs ( RAP1B, GSE1, CORO1C, PCMTD1 and Plt-circR4 ) and for one linear mRNA control ( RAP1B ). The positions of ribosomal RNAs are marked by arrowheads (5S, 18S and 28S). M , markers (300, 200, 100 bp); lane I, 10% platelet extract input. The right panel shows quantification of the gradient fractionation of extract vs. RNA.
    Figure Legend Snippet: CircRNA-protein complexes in human platelets. Sedimentation profiles of circRNPs. Platelet extract and corresponding free RNA (prepared from the same extract) were fractionated by glycerol gradient centrifugation (fractions #1–12 from top to bottom), followed by RT-PCR analysis across the gradient for five abundant circRNAs ( RAP1B, GSE1, CORO1C, PCMTD1 and Plt-circR4 ) and for one linear mRNA control ( RAP1B ). The positions of ribosomal RNAs are marked by arrowheads (5S, 18S and 28S). M , markers (300, 200, 100 bp); lane I, 10% platelet extract input. The right panel shows quantification of the gradient fractionation of extract vs. RNA.

    Techniques Used: Sedimentation, Gradient Centrifugation, Reverse Transcription Polymerase Chain Reaction, Fractionation

    16) Product Images from "Optimizing exosomal RNA isolation for RNA-Seq analyses of archival sera specimens"

    Article Title: Optimizing exosomal RNA isolation for RNA-Seq analyses of archival sera specimens

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0196913

    Flow chart for evaluation of exosomal RNAs from cell-free sera as biomarkers for human diseases. Graphic summary of the workflow including time allotment for preparation for cell-free serum (steps ①-③), comparison of methods for exosome enrichment (step ④), validation by transmission electron microscopy (TEM) and immunoblotting for CD63 or other exosomal markers (step ⑤), RNA extraction (step ⑥), and preparation of RNA-Seq libraries (step ⑦). 10–100 nanograms RNA can be used for library preparation with the NEBNext Ultra Directional RNA Library Prep Kit. Step ② is an optional centrifugation step that can be included to ensure the most efficient removal of trace amounts of cell debris and shedding microvesicles. A validation step can be performed with RT-qPCR for specific candidate RNA following RNA-Seq analysis (step ⑧).
    Figure Legend Snippet: Flow chart for evaluation of exosomal RNAs from cell-free sera as biomarkers for human diseases. Graphic summary of the workflow including time allotment for preparation for cell-free serum (steps ①-③), comparison of methods for exosome enrichment (step ④), validation by transmission electron microscopy (TEM) and immunoblotting for CD63 or other exosomal markers (step ⑤), RNA extraction (step ⑥), and preparation of RNA-Seq libraries (step ⑦). 10–100 nanograms RNA can be used for library preparation with the NEBNext Ultra Directional RNA Library Prep Kit. Step ② is an optional centrifugation step that can be included to ensure the most efficient removal of trace amounts of cell debris and shedding microvesicles. A validation step can be performed with RT-qPCR for specific candidate RNA following RNA-Seq analysis (step ⑧).

    Techniques Used: Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, RNA Extraction, RNA Sequencing Assay, Centrifugation, Quantitative RT-PCR

    17) Product Images from "Spatially clustered loci with multiple enhancers are frequent targets of HIV-1 integration"

    Article Title: Spatially clustered loci with multiple enhancers are frequent targets of HIV-1 integration

    Journal: Nature Communications

    doi: 10.1038/s41467-019-12046-3

    HIV-1 integration hotspots are within genes proximal to super-enhancers (SEs). a Metagene plots of H3K27ac, H3K4me1, H3K4me3, BRD4, MED1, H3K36me3, H4K20me1, H3K9me2, and H3K27me3 ChIP-Seq signals in recurrent integration genes (RIGs), which are protein coding in red and the rest of the protein-coding genes that are not targeted by HIV-1 (no RIGs) in black. b ROC analysis represented in heatmap summarizing the co-occurrence of integration sites and epigenetic modification obtained by ChIP-Seq for H3K27ac, H3K4me1, BRD4, MED1, H3K36me3, H4K20me1, H3K4me3, H3K27me3, and H3K9me2. HIV-1 integration datasets are shown in the columns, and epigenetic modifications are shown in rows. Associations are quantified using the ROC area method; values of ROC areas are shown in the color key at the right. c Distance to the nearest SE in activated CD4 + T cells. Box plots represent distances from the gene to the nearest SE grouped by number of times the gene is found in different datasets. d FOXP1 , STAT5B , and BACH2 IS (black) superimposition on H3K27ac (orange), SE (blue), H3K36me3 (green), and BRD4 (violet) ChIP-Seq tracks
    Figure Legend Snippet: HIV-1 integration hotspots are within genes proximal to super-enhancers (SEs). a Metagene plots of H3K27ac, H3K4me1, H3K4me3, BRD4, MED1, H3K36me3, H4K20me1, H3K9me2, and H3K27me3 ChIP-Seq signals in recurrent integration genes (RIGs), which are protein coding in red and the rest of the protein-coding genes that are not targeted by HIV-1 (no RIGs) in black. b ROC analysis represented in heatmap summarizing the co-occurrence of integration sites and epigenetic modification obtained by ChIP-Seq for H3K27ac, H3K4me1, BRD4, MED1, H3K36me3, H4K20me1, H3K4me3, H3K27me3, and H3K9me2. HIV-1 integration datasets are shown in the columns, and epigenetic modifications are shown in rows. Associations are quantified using the ROC area method; values of ROC areas are shown in the color key at the right. c Distance to the nearest SE in activated CD4 + T cells. Box plots represent distances from the gene to the nearest SE grouped by number of times the gene is found in different datasets. d FOXP1 , STAT5B , and BACH2 IS (black) superimposition on H3K27ac (orange), SE (blue), H3K36me3 (green), and BRD4 (violet) ChIP-Seq tracks

    Techniques Used: Chromatin Immunoprecipitation, Modification

    18) Product Images from "Advanced Safety and Genetic Stability in Mice of a Novel DNA-Launched Venezuelan Equine Encephalitis Virus Vaccine with Rearranged Structural Genes"

    Article Title: Advanced Safety and Genetic Stability in Mice of a Novel DNA-Launched Venezuelan Equine Encephalitis Virus Vaccine with Rearranged Structural Genes

    Journal: Vaccines

    doi: 10.3390/vaccines8010114

    RNA-Seq by Illumina NextSeq was run on rRNA depleted RNA isolated from brain tissue of mice receiving the passaged virus on day 2 post IC inoculation. Sequences were quality trimmed with trimmomatic and aligned to VEEV TC-83 or V4020 reference sequences with BowTie2. Pile-ups were generated and filtered in Galaxy before being exported to excel to calculate normalized read depth ( a ), statistical significance of mutations ( a ), mutation frequency ( b ) and SMR ( c ). ( a ) Normalized read depth plotted on the left y-axis (red for V4020, purple for TC-83) was calculated by dividing the number of virus-aligned reads at each position by the sum of the number of reads for every position. Statistical significance of mutation rate as a Z-score, plotted by nucleotide position on the right y-axis (blue for V4020 and grey for TC-83), was calculated in Excel using the “NORM.DIST” function to calculate a probability density function based on the mean and standard deviation of each biological sample. The boundaries between genes and certain features are marked with a dashed vertical line and labeled at the bottom of the panel. Z-scores over 10 −25 are reported as this value. Passages 2–5 of V4020 and Passage 1 of TC-83 are the average of two biological replicates. All other data are representative of a single biological replicate. ( b ) Mutation frequency calculated as a ratio of a mutation occurring at the identified location divided by the total number of reads for each reported location for V4020 (blue) and TC-83 (red). ( c ) SMR for the overall coding sequences and individual genes of V4020 (blue) and TC-83 (red). * p
    Figure Legend Snippet: RNA-Seq by Illumina NextSeq was run on rRNA depleted RNA isolated from brain tissue of mice receiving the passaged virus on day 2 post IC inoculation. Sequences were quality trimmed with trimmomatic and aligned to VEEV TC-83 or V4020 reference sequences with BowTie2. Pile-ups were generated and filtered in Galaxy before being exported to excel to calculate normalized read depth ( a ), statistical significance of mutations ( a ), mutation frequency ( b ) and SMR ( c ). ( a ) Normalized read depth plotted on the left y-axis (red for V4020, purple for TC-83) was calculated by dividing the number of virus-aligned reads at each position by the sum of the number of reads for every position. Statistical significance of mutation rate as a Z-score, plotted by nucleotide position on the right y-axis (blue for V4020 and grey for TC-83), was calculated in Excel using the “NORM.DIST” function to calculate a probability density function based on the mean and standard deviation of each biological sample. The boundaries between genes and certain features are marked with a dashed vertical line and labeled at the bottom of the panel. Z-scores over 10 −25 are reported as this value. Passages 2–5 of V4020 and Passage 1 of TC-83 are the average of two biological replicates. All other data are representative of a single biological replicate. ( b ) Mutation frequency calculated as a ratio of a mutation occurring at the identified location divided by the total number of reads for each reported location for V4020 (blue) and TC-83 (red). ( c ) SMR for the overall coding sequences and individual genes of V4020 (blue) and TC-83 (red). * p

    Techniques Used: RNA Sequencing Assay, Isolation, Mouse Assay, Generated, Mutagenesis, Standard Deviation, Labeling

    19) Product Images from "Sensory neuron lineage mapping and manipulation in the Drosophila olfactory system"

    Article Title: Sensory neuron lineage mapping and manipulation in the Drosophila olfactory system

    Journal: Nature Communications

    doi: 10.1038/s41467-019-08345-4

    Loss of Pnt results in Naa-to-Nab transformations in diverse sensillar subtypes. a A sensillum can contain up to four OSNs through differentiation of Naa (cyan), Nab (magenta), Nba (green), Nbb (yellow) terminal daughter cells originating from a single SOP lineage. b Representative images of RNA FISH for Or67d (magenta) in at1 sensilla in control and pnt RNAi antennae. In pnt RNAi antennae, Or67d-expressing OSNs are duplicated (arrow). A schematic of the proposed Naa-to-Nab fate transformation is shown on the right (color scheme as in ( a )). Scale bar = 2 µm. The open circles in this and other schematics represent OSN precursors that have undergone apoptosis. c Representative images of RNA FISH for Or85a (magenta) and Or59b (green) in ab2 sensilla in control and pnt RNAi antennae. In pnt RNAi antennae, Or85a OSNs (Nab) are duplicated (arrow), while Or59b OSNs (Nba) are unaffected. d Representative images of RNA FISH for Or85b (magenta) and Or22a (green) in ab3 sensilla in control and pnt RNAi antennae. In pnt RNAi antennae, Or85b OSNs (Nab) are duplicated (arrow), while Or22a OSNs (Nba) are unaffected. e Representative images of RNA FISH for Or92a (magenta) and Or10a (cyan) in ab1 sensilla in control and pnt RNAi antennae. In pnt RNAi antennae, Or92a OSNs (Nab) are duplicated (arrow), while Or10a OSNs (Naa) are lost. f Top: theoretical ratios of OSN types in 2-, 3- and 4-neuron sensilla in control and pnt RNAi antennae, assuming Naa-to-Nab fate transformation (i.e. loss of Naa OSNs, and duplication of Nab OSNs). Bottom: experimentally determined OSN ratios in all sensilla in pnt RNAi antennae using as a proxy the normalized ratios of olfactory receptor mRNA expression from antennal transcriptomes (see Supplementary Fig. 6e ). In ab10, Or49a is reported to be coexpressed with Or85f 13 , but transcript levels for this gene were below the cut-off applied during the analysis of these RNA-seq datasets
    Figure Legend Snippet: Loss of Pnt results in Naa-to-Nab transformations in diverse sensillar subtypes. a A sensillum can contain up to four OSNs through differentiation of Naa (cyan), Nab (magenta), Nba (green), Nbb (yellow) terminal daughter cells originating from a single SOP lineage. b Representative images of RNA FISH for Or67d (magenta) in at1 sensilla in control and pnt RNAi antennae. In pnt RNAi antennae, Or67d-expressing OSNs are duplicated (arrow). A schematic of the proposed Naa-to-Nab fate transformation is shown on the right (color scheme as in ( a )). Scale bar = 2 µm. The open circles in this and other schematics represent OSN precursors that have undergone apoptosis. c Representative images of RNA FISH for Or85a (magenta) and Or59b (green) in ab2 sensilla in control and pnt RNAi antennae. In pnt RNAi antennae, Or85a OSNs (Nab) are duplicated (arrow), while Or59b OSNs (Nba) are unaffected. d Representative images of RNA FISH for Or85b (magenta) and Or22a (green) in ab3 sensilla in control and pnt RNAi antennae. In pnt RNAi antennae, Or85b OSNs (Nab) are duplicated (arrow), while Or22a OSNs (Nba) are unaffected. e Representative images of RNA FISH for Or92a (magenta) and Or10a (cyan) in ab1 sensilla in control and pnt RNAi antennae. In pnt RNAi antennae, Or92a OSNs (Nab) are duplicated (arrow), while Or10a OSNs (Naa) are lost. f Top: theoretical ratios of OSN types in 2-, 3- and 4-neuron sensilla in control and pnt RNAi antennae, assuming Naa-to-Nab fate transformation (i.e. loss of Naa OSNs, and duplication of Nab OSNs). Bottom: experimentally determined OSN ratios in all sensilla in pnt RNAi antennae using as a proxy the normalized ratios of olfactory receptor mRNA expression from antennal transcriptomes (see Supplementary Fig. 6e ). In ab10, Or49a is reported to be coexpressed with Or85f 13 , but transcript levels for this gene were below the cut-off applied during the analysis of these RNA-seq datasets

    Techniques Used: Fluorescence In Situ Hybridization, Expressing, Transformation Assay, RNA Sequencing Assay

    An OSN lineage-specific driver. a Top row: developmental expression of the nonimmortalized GMR82D08-GAL4 (hereafter, at1 driver) using a myr:GFP reporter (green) in the antennal disc SOPs (region marked by α-Dac (blue)) during late larval/early pupal stages. Bottom row: the at1 driver is expressed in the daughter cells of these SOPs in the developing pupal antenna but progressively loses its expression from 20 h APF as OSNs differentiate (visualized with the neuronal marker α-Elav (magenta)). Scale bar = 20 µm in this and other panels. b Immortalization of the at1 driver reveals labeling of clusters of cells in the adult antenna by an rCD2:GFP reporter (green). RNA fluorescence in situ hybridization demonstrates that a single cell within each cluster (arrowheads in the inset images) expresses Or67d mRNA (magenta). c Representative example of a single sensillum in the adult antenna labeled by the immortalized at1 driver, viewed at three focal planes. There is a single Or67d mRNA-positive OSN (cell 1, arrowhead), flanked by four non-neuronal support cells (cells 2–5). d Sensilla cells labeled by the immortalized at1 driver lineage (α-GFP; green) also express Lush (magenta), an odorant binding protein unique to trichoid sensilla support cells 72
    Figure Legend Snippet: An OSN lineage-specific driver. a Top row: developmental expression of the nonimmortalized GMR82D08-GAL4 (hereafter, at1 driver) using a myr:GFP reporter (green) in the antennal disc SOPs (region marked by α-Dac (blue)) during late larval/early pupal stages. Bottom row: the at1 driver is expressed in the daughter cells of these SOPs in the developing pupal antenna but progressively loses its expression from 20 h APF as OSNs differentiate (visualized with the neuronal marker α-Elav (magenta)). Scale bar = 20 µm in this and other panels. b Immortalization of the at1 driver reveals labeling of clusters of cells in the adult antenna by an rCD2:GFP reporter (green). RNA fluorescence in situ hybridization demonstrates that a single cell within each cluster (arrowheads in the inset images) expresses Or67d mRNA (magenta). c Representative example of a single sensillum in the adult antenna labeled by the immortalized at1 driver, viewed at three focal planes. There is a single Or67d mRNA-positive OSN (cell 1, arrowhead), flanked by four non-neuronal support cells (cells 2–5). d Sensilla cells labeled by the immortalized at1 driver lineage (α-GFP; green) also express Lush (magenta), an odorant binding protein unique to trichoid sensilla support cells 72

    Techniques Used: Expressing, Marker, Labeling, Fluorescence, In Situ Hybridization, Binding Assay

    20) Product Images from "Sensory neuron lineage mapping and manipulation in the Drosophila olfactory system"

    Article Title: Sensory neuron lineage mapping and manipulation in the Drosophila olfactory system

    Journal: Nature Communications

    doi: 10.1038/s41467-019-08345-4

    An OSN lineage-specific driver. a Top row: developmental expression of the nonimmortalized GMR82D08-GAL4 (hereafter, at1 driver) using a myr:GFP reporter (green) in the antennal disc SOPs (region marked by α-Dac (blue)) during late larval/early pupal stages. Bottom row: the at1 driver is expressed in the daughter cells of these SOPs in the developing pupal antenna but progressively loses its expression from 20 h APF as OSNs differentiate (visualized with the neuronal marker α-Elav (magenta)). Scale bar = 20 µm in this and other panels. b Immortalization of the at1 driver reveals labeling of clusters of cells in the adult antenna by an rCD2:GFP reporter (green). RNA fluorescence in situ hybridization demonstrates that a single cell within each cluster (arrowheads in the inset images) expresses Or67d mRNA (magenta). c Representative example of a single sensillum in the adult antenna labeled by the immortalized at1 driver, viewed at three focal planes. There is a single Or67d mRNA-positive OSN (cell 1, arrowhead), flanked by four non-neuronal support cells (cells 2–5). d
    Figure Legend Snippet: An OSN lineage-specific driver. a Top row: developmental expression of the nonimmortalized GMR82D08-GAL4 (hereafter, at1 driver) using a myr:GFP reporter (green) in the antennal disc SOPs (region marked by α-Dac (blue)) during late larval/early pupal stages. Bottom row: the at1 driver is expressed in the daughter cells of these SOPs in the developing pupal antenna but progressively loses its expression from 20 h APF as OSNs differentiate (visualized with the neuronal marker α-Elav (magenta)). Scale bar = 20 µm in this and other panels. b Immortalization of the at1 driver reveals labeling of clusters of cells in the adult antenna by an rCD2:GFP reporter (green). RNA fluorescence in situ hybridization demonstrates that a single cell within each cluster (arrowheads in the inset images) expresses Or67d mRNA (magenta). c Representative example of a single sensillum in the adult antenna labeled by the immortalized at1 driver, viewed at three focal planes. There is a single Or67d mRNA-positive OSN (cell 1, arrowhead), flanked by four non-neuronal support cells (cells 2–5). d

    Techniques Used: Expressing, Marker, Labeling, Fluorescence, In Situ Hybridization

    21) Product Images from "Probing the sRNA regulatory landscape of P. aeruginosa: post-transcriptional control of determinants of pathogenicity and antibiotic susceptibility"

    Article Title: Probing the sRNA regulatory landscape of P. aeruginosa: post-transcriptional control of determinants of pathogenicity and antibiotic susceptibility

    Journal: Molecular microbiology

    doi: 10.1111/mmi.13857

    Schematic overview of Hi-GRIL-seq. Induction of T4 RNA ligase expression from the P tac promoter with IPTG leads to the expression of the enzyme and the formation of chimeras between base paired endogenous sRNAs and their targets. Following isolation of total RNA and rRNA depletion, a cDNA library for Illumina sequencing is constructed and sequenced. RNA interactions between sRNAs and their targets are identified by a BLAST-based analysis pipeline. Global chimeras are visualized in a two-dimensional dot plot, in which the location of the dot represents the genomic coordinate of the participating RNAs. To examine the targets of a particular RNA, the coverage of its targets can be visualized. To further zoom in on a particular interaction between the two RNAs, the exact location of ligation junctions in the chimeras are mapped and visualized.
    Figure Legend Snippet: Schematic overview of Hi-GRIL-seq. Induction of T4 RNA ligase expression from the P tac promoter with IPTG leads to the expression of the enzyme and the formation of chimeras between base paired endogenous sRNAs and their targets. Following isolation of total RNA and rRNA depletion, a cDNA library for Illumina sequencing is constructed and sequenced. RNA interactions between sRNAs and their targets are identified by a BLAST-based analysis pipeline. Global chimeras are visualized in a two-dimensional dot plot, in which the location of the dot represents the genomic coordinate of the participating RNAs. To examine the targets of a particular RNA, the coverage of its targets can be visualized. To further zoom in on a particular interaction between the two RNAs, the exact location of ligation junctions in the chimeras are mapped and visualized.

    Techniques Used: Expressing, Isolation, cDNA Library Assay, Sequencing, Construct, Ligation

    22) Product Images from "Heterochromatin-associated interactions of Drosophila HP1a with dADD1, HIPP1, and repetitive RNAs"

    Article Title: Heterochromatin-associated interactions of Drosophila HP1a with dADD1, HIPP1, and repetitive RNAs

    Journal: Genes & Development

    doi: 10.1101/gad.241950.114

    RNA-seq analysis of BioTAP-XL pull-downs. ( A ) Enrichment of repeat-derived RNA in HP1a-BioTAP cross-linked complexes from S2 cells compared with MSL3-BioTAP complexes from S2 cells detected using a random-priming approach for cDNA synthesis and Illumina
    Figure Legend Snippet: RNA-seq analysis of BioTAP-XL pull-downs. ( A ) Enrichment of repeat-derived RNA in HP1a-BioTAP cross-linked complexes from S2 cells compared with MSL3-BioTAP complexes from S2 cells detected using a random-priming approach for cDNA synthesis and Illumina

    Techniques Used: RNA Sequencing Assay, Derivative Assay

    23) Product Images from "Transgenerational Effects of Extended Dauer Diapause on Starvation Survival and Gene Expression Plasticity in Caenorhabditis elegans"

    Article Title: Transgenerational Effects of Extended Dauer Diapause on Starvation Survival and Gene Expression Plasticity in Caenorhabditis elegans

    Journal: Genetics

    doi: 10.1534/genetics.118.301250

    mRNA-seq reveals relative contributions of current environment and ancestral environment in shaping gene expression variation. (A) Schematic of experimental set-up for collection of RNA-seq samples. (B) Principal component analysis (PCA) of six conditions including all 8649 reliably detected genes; 97% of variance explained by whether worms were fed or starved at collection (PC1), and 1.3% of variance explained by whether ancestors experienced control, short-term dauer, or long-term dauer conditions (PC2). Mean CPM of biological replicates were used for each condition for PCA. Number of biological replicates: control starved (12), ST dauer starved (9), LT dauer starved (6), control fed (4), ST dauer fed (4), and LT dauer fed (3).
    Figure Legend Snippet: mRNA-seq reveals relative contributions of current environment and ancestral environment in shaping gene expression variation. (A) Schematic of experimental set-up for collection of RNA-seq samples. (B) Principal component analysis (PCA) of six conditions including all 8649 reliably detected genes; 97% of variance explained by whether worms were fed or starved at collection (PC1), and 1.3% of variance explained by whether ancestors experienced control, short-term dauer, or long-term dauer conditions (PC2). Mean CPM of biological replicates were used for each condition for PCA. Number of biological replicates: control starved (12), ST dauer starved (9), LT dauer starved (6), control fed (4), ST dauer fed (4), and LT dauer fed (3).

    Techniques Used: Expressing, RNA Sequencing Assay

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky603

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

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

    25) Product Images from "m6A enhances the phase separation potential of mRNA"

    Article Title: m6A enhances the phase separation potential of mRNA

    Journal: Nature

    doi: 10.1038/s41586-019-1374-1

    Confirmation of the Mettl14 knockout model and DF2 phase-separation into P-bodies in mES cells a , Mettl14 knockout (KO) mES cells are depleted in m 6 A RNA. We sought to independently confirm the depletion of m 6 A from mRNA in these cells, which were previously shown to have 99% reduction in m 6 A 19 . The TLC assay selectively quantifies m 6 A in a G-A-C context, thereby reducing the possibility of contamination of m 6 A from rRNA or snRNA, which are in a A-A-C or C-A-G context, respectively 38 . The protocol was performed as described previously 38 . Indicated in the TLC chromatograms is the relative position of m 6 A (dotted circle) and adenosine (A), cytosine (C), and uracil (U). Left and the right panels show radiochromatograms obtained from 2D-TLC of poly(A) RNA from wild-type and Mettl14 knockout cells. No m 6 A is detectable in the poly(A) RNA derived from Mettl14 knockout cells confirming the efficiency of m 6 A depletion in these cells. Experiments were performed in duplicate. mES cells are used here since m 6 A depletion can be readily achieved in Mettl14 knockout mES cells without impairing viability 19 . In contrast, m 6 A depletion cannot be readily achieved in immortalized cell lines as both Mettl3 and Mettl14 are essential for nearly all cell lines 52 . b , DF2 partitioning into stress granules induced by arsenite is impaired in m 6 A-deficient cells. This impairment is similar to that shown in stress granules induced by heat shock as seen in Figure 3a . Experiment was performed in triplicate.
    Figure Legend Snippet: Confirmation of the Mettl14 knockout model and DF2 phase-separation into P-bodies in mES cells a , Mettl14 knockout (KO) mES cells are depleted in m 6 A RNA. We sought to independently confirm the depletion of m 6 A from mRNA in these cells, which were previously shown to have 99% reduction in m 6 A 19 . The TLC assay selectively quantifies m 6 A in a G-A-C context, thereby reducing the possibility of contamination of m 6 A from rRNA or snRNA, which are in a A-A-C or C-A-G context, respectively 38 . The protocol was performed as described previously 38 . Indicated in the TLC chromatograms is the relative position of m 6 A (dotted circle) and adenosine (A), cytosine (C), and uracil (U). Left and the right panels show radiochromatograms obtained from 2D-TLC of poly(A) RNA from wild-type and Mettl14 knockout cells. No m 6 A is detectable in the poly(A) RNA derived from Mettl14 knockout cells confirming the efficiency of m 6 A depletion in these cells. Experiments were performed in duplicate. mES cells are used here since m 6 A depletion can be readily achieved in Mettl14 knockout mES cells without impairing viability 19 . In contrast, m 6 A depletion cannot be readily achieved in immortalized cell lines as both Mettl3 and Mettl14 are essential for nearly all cell lines 52 . b , DF2 partitioning into stress granules induced by arsenite is impaired in m 6 A-deficient cells. This impairment is similar to that shown in stress granules induced by heat shock as seen in Figure 3a . Experiment was performed in triplicate.

    Techniques Used: Knock-Out, Thin Layer Chromatography, Derivative Assay

    26) Product Images from "Cooperative molecular networks drive a mammalian cell state transition"

    Article Title: Cooperative molecular networks drive a mammalian cell state transition

    Journal: bioRxiv

    doi: 10.1101/2020.03.23.000109

    (A) The top 15 candidate genes from the haploid screen ordered by the number of times the hit was found in the 35 screens, including the number of independent integration sites and the calculated FDR. (B) Significantly enriched pathways among screen hits ranked according to fold enrichment; colours represent FDR in screen analysis. Numbers next to bars indicate number of hit-genes within category, (C) Significantly enriched GO terms among screen hits as in (B) (D) Workflow to generate Cas9 knockouts in RC9 ESCs. (E) Idealized strategy illustrating gRNAs and genotyping primers. (F) Western analysis using indicated KOs and indicated antibodies. Tubulin or Gapdh were used as loading controls, as indicated. (G) Anti-flag specific Westerns in indicated KO rescue ESCs upon stable forced expression of 3xflag rescue cDNAs driven from a CAG promoter. (H) Rex1GFP levels measured by FACS showing restoration of differentiation behaviour in the indicated rescue cell lines at N30. (I) t-SNE visualization of RNA-seq profiles in 2i and at N24 before and after batch correction. (J) GO enrichment analysis of the top 10% of genes (by absolute logFC) that were differentially expressed between 2i and N24 in WT ESCs (FDR ≤ 0.05, H0: |FC|
    Figure Legend Snippet: (A) The top 15 candidate genes from the haploid screen ordered by the number of times the hit was found in the 35 screens, including the number of independent integration sites and the calculated FDR. (B) Significantly enriched pathways among screen hits ranked according to fold enrichment; colours represent FDR in screen analysis. Numbers next to bars indicate number of hit-genes within category, (C) Significantly enriched GO terms among screen hits as in (B) (D) Workflow to generate Cas9 knockouts in RC9 ESCs. (E) Idealized strategy illustrating gRNAs and genotyping primers. (F) Western analysis using indicated KOs and indicated antibodies. Tubulin or Gapdh were used as loading controls, as indicated. (G) Anti-flag specific Westerns in indicated KO rescue ESCs upon stable forced expression of 3xflag rescue cDNAs driven from a CAG promoter. (H) Rex1GFP levels measured by FACS showing restoration of differentiation behaviour in the indicated rescue cell lines at N30. (I) t-SNE visualization of RNA-seq profiles in 2i and at N24 before and after batch correction. (J) GO enrichment analysis of the top 10% of genes (by absolute logFC) that were differentially expressed between 2i and N24 in WT ESCs (FDR ≤ 0.05, H0: |FC|

    Techniques Used: Western Blot, Expressing, FACS, RNA Sequencing Assay

    27) Product Images from "MicroRNAs Circulate in the Hemolymph of Drosophila and Accumulate Relative to Tissue microRNAs in an Age-Dependent Manner"

    Article Title: MicroRNAs Circulate in the Hemolymph of Drosophila and Accumulate Relative to Tissue microRNAs in an Age-Dependent Manner

    Journal: Genomics Insights

    doi: 10.4137/GEI.S38147

    Presence of stable miRNAs in Drosophila melanogaster hemolymph. ( A , B ) Clear HL droplets extruded from fly head and thorax. ( C – F ) Real-time qPCR amplification of selected HL miRNAs and mRNAs. Total RNA including small RNA was extracted from HL samples and analyzed with qPCR to measure the levels of miRNAs and mRNAs. The y -axis represents the relative fluorescence units (RFU) in a semi-log scale. The x -axis represents the cycle at which fluorescence was detected above an automatically determined threshold. ( C ) Amplification plots for miR-14, miR-8, and miR-184 measured in a representative HL sample. ( D ) Amplification plots for miR-14, let-7, bantam, and spiked-in synthetic C. elegans cel-miR-39 RNA in representative HL sample. ( E ) The amplification plots for tubulin, actin, and gapdh mRNAs determined by qPCR using total RNA from HL and S2 cells. Sample from S2 cells are used as a positive control for detecting Drosophila mRNAs by qPCR. The amplification curves of all three mRNAs are superimposed on one another, reflecting the presence of similar amounts of these mRNA in S2 cells. Amplification curves from HL samples show that fluorescent products appear after about 30 cycles, reflecting the significantly lower abundance of these mRNAs in HL relative to S2 cells. ( F ) The cycle threshold (Ct) fold-change of selected miRNA amplified in the absence or presence of RNase A and DNase I. Total RNA was extracted from HL samples spiked with 10 fmoles of cel-miR-39 RNA. The x -axis represents the ratio of raw Ct values from control samples divided by raw Ct values from samples incubated with RNase A and DNase I. The significantly higher magnitude of the Ct fold change of the spiked-in synthetic miRNA relative to those of the miRNA indicates that the HL-miRNA are present in nuclease-resistant, stable form.
    Figure Legend Snippet: Presence of stable miRNAs in Drosophila melanogaster hemolymph. ( A , B ) Clear HL droplets extruded from fly head and thorax. ( C – F ) Real-time qPCR amplification of selected HL miRNAs and mRNAs. Total RNA including small RNA was extracted from HL samples and analyzed with qPCR to measure the levels of miRNAs and mRNAs. The y -axis represents the relative fluorescence units (RFU) in a semi-log scale. The x -axis represents the cycle at which fluorescence was detected above an automatically determined threshold. ( C ) Amplification plots for miR-14, miR-8, and miR-184 measured in a representative HL sample. ( D ) Amplification plots for miR-14, let-7, bantam, and spiked-in synthetic C. elegans cel-miR-39 RNA in representative HL sample. ( E ) The amplification plots for tubulin, actin, and gapdh mRNAs determined by qPCR using total RNA from HL and S2 cells. Sample from S2 cells are used as a positive control for detecting Drosophila mRNAs by qPCR. The amplification curves of all three mRNAs are superimposed on one another, reflecting the presence of similar amounts of these mRNA in S2 cells. Amplification curves from HL samples show that fluorescent products appear after about 30 cycles, reflecting the significantly lower abundance of these mRNAs in HL relative to S2 cells. ( F ) The cycle threshold (Ct) fold-change of selected miRNA amplified in the absence or presence of RNase A and DNase I. Total RNA was extracted from HL samples spiked with 10 fmoles of cel-miR-39 RNA. The x -axis represents the ratio of raw Ct values from control samples divided by raw Ct values from samples incubated with RNase A and DNase I. The significantly higher magnitude of the Ct fold change of the spiked-in synthetic miRNA relative to those of the miRNA indicates that the HL-miRNA are present in nuclease-resistant, stable form.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Fluorescence, Positive Control, Incubation

    Outline of the experimental design and workflow for the integrated analysis of the miRNA-Seq and mRNA-Seq data. Genes that are potentially regulated by the HL-miRNAs were determined by integrating miRNA-Seq and mRNA-Seq data using the following three steps: Step 1 (depicted in green): After identifying the HL-miRNAs enriched relative to BT in either young or old, or both young and old ( Tables 1 and 2 ), we retrieved their computationally predicted target genes from the m 3 RNA database ( http://m3rna.cnb.csic.es ). Step 2 (depicted in blue): mRNA-Seq was utilized to identify three groups of genes that were differentially expressed with age, either upregulated, downregulated, or unchanged in BT. Step 3 (depicted in red): The three groups of genes predicted in step1 as targets of HL-miRNAs were intersected with the three groups of genes identified in step 2. In the figure, ( A ) depicts the derivation of genes that are predicted to be targets of HL-miRNAs enriched in young which are also upregulated with age in BT. These genes (Supplementary Table 1 ) are likely upregulated in BT of old flies because their expression is no longer repressed by HL-miRNAs that are enriched only in young flies. ( B ) Depicts the derivation of genes which are the predicted to be targets of HL-miRNAs enriched in old which are also downregulated with age in BT. These genes (Supplementary Table 2 ) are likely downregulated in BT of old flies because their expression is repressed by HL-miRNAs which are enriched only in old flies. ( C ) Depicts the derivation of genes which are predicted to be targets of HL-miRNAs enriched in both young and old which also do not change expression with age in BT. These genes (Supplementary Table 3) do not change expression with age because the concentration of their regulatory miRNAs in HL does not change with age.
    Figure Legend Snippet: Outline of the experimental design and workflow for the integrated analysis of the miRNA-Seq and mRNA-Seq data. Genes that are potentially regulated by the HL-miRNAs were determined by integrating miRNA-Seq and mRNA-Seq data using the following three steps: Step 1 (depicted in green): After identifying the HL-miRNAs enriched relative to BT in either young or old, or both young and old ( Tables 1 and 2 ), we retrieved their computationally predicted target genes from the m 3 RNA database ( http://m3rna.cnb.csic.es ). Step 2 (depicted in blue): mRNA-Seq was utilized to identify three groups of genes that were differentially expressed with age, either upregulated, downregulated, or unchanged in BT. Step 3 (depicted in red): The three groups of genes predicted in step1 as targets of HL-miRNAs were intersected with the three groups of genes identified in step 2. In the figure, ( A ) depicts the derivation of genes that are predicted to be targets of HL-miRNAs enriched in young which are also upregulated with age in BT. These genes (Supplementary Table 1 ) are likely upregulated in BT of old flies because their expression is no longer repressed by HL-miRNAs that are enriched only in young flies. ( B ) Depicts the derivation of genes which are the predicted to be targets of HL-miRNAs enriched in old which are also downregulated with age in BT. These genes (Supplementary Table 2 ) are likely downregulated in BT of old flies because their expression is repressed by HL-miRNAs which are enriched only in old flies. ( C ) Depicts the derivation of genes which are predicted to be targets of HL-miRNAs enriched in both young and old which also do not change expression with age in BT. These genes (Supplementary Table 3) do not change expression with age because the concentration of their regulatory miRNAs in HL does not change with age.

    Techniques Used: Expressing, Concentration Assay

    28) Product Images from "Recognition of RNA N6-methyladenosine by IGF2BP Proteins Enhances mRNA Stability and Translation"

    Article Title: Recognition of RNA N6-methyladenosine by IGF2BP Proteins Enhances mRNA Stability and Translation

    Journal: Nature cell biology

    doi: 10.1038/s41556-018-0045-z

    The KH domains of IGF2BPs are critical for m 6 A recognition and binding ( a ) Schematic structures showing RNA binding domains within IGF2BP proteins and a summary of IGF2BP variants used in this study. Blue boxes are RRM domains, red boxes are wild-type KH domains with GxxG core, and grey boxes are inactive KH domain with GxxG to GEEG conversions. ( b ) RNA pulldown followed by Western blotting showed in vitro binding of ssRNA baits with wild-type (wt) or KH domain-mutated IGF2BP variants, representative of 3 independent experiments. ( c ) In vitro binding of CRD1 RNA probes with wild-type or KH3-4 mutated IGF2BPs, representative of 3 independent experiments. ( d ) The association of wild-type and KH3-4 mutated IGF2BPs with MYC CRD in HEK293T cells as assessed by RIP-qPCR. ( e ) Relative luciferase activity of CRD reporters in HEK293T cells with forced expression of wild-type or mutated IGF2BP2 variants. ( f ) Changes in MYC mRNA levels in Hela cells with empty vector or forced expression of wild-type or KH3-4 mutated IGF2BPs one hour post-heat shock (HS). Values are mean±s.d. of n =3 independent experiments, and two-tailed Student’s t -tests were used in d , e , f (*, P
    Figure Legend Snippet: The KH domains of IGF2BPs are critical for m 6 A recognition and binding ( a ) Schematic structures showing RNA binding domains within IGF2BP proteins and a summary of IGF2BP variants used in this study. Blue boxes are RRM domains, red boxes are wild-type KH domains with GxxG core, and grey boxes are inactive KH domain with GxxG to GEEG conversions. ( b ) RNA pulldown followed by Western blotting showed in vitro binding of ssRNA baits with wild-type (wt) or KH domain-mutated IGF2BP variants, representative of 3 independent experiments. ( c ) In vitro binding of CRD1 RNA probes with wild-type or KH3-4 mutated IGF2BPs, representative of 3 independent experiments. ( d ) The association of wild-type and KH3-4 mutated IGF2BPs with MYC CRD in HEK293T cells as assessed by RIP-qPCR. ( e ) Relative luciferase activity of CRD reporters in HEK293T cells with forced expression of wild-type or mutated IGF2BP2 variants. ( f ) Changes in MYC mRNA levels in Hela cells with empty vector or forced expression of wild-type or KH3-4 mutated IGF2BPs one hour post-heat shock (HS). Values are mean±s.d. of n =3 independent experiments, and two-tailed Student’s t -tests were used in d , e , f (*, P

    Techniques Used: Binding Assay, RNA Binding Assay, Western Blot, In Vitro, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Expressing, Plasmid Preparation, Two Tailed Test

    IGF2BPs regulate MYC expression through binding to methylated CRD ( a ) Distribution of m 6 A peaks across MYC mRNA transcript. The coding region instability determinant (CRD) region is highlighted in yellow. m 6 A-seq was repeated twice while RIP-seq was performed once.( b ) RIP-qPCR showing the association of MYC CRD with FLAG-tagged IGF2BPs in HEK293T cells. ( c ) Enrichment of m 6 A modification in MYC CRD as detected by gene specific m 6 A qPCR assay. ( d ) RIP-qPCR showing the binding of METTL3 and METTL14 to the MYC CRD. ( e ) RNA pulldown of endogenous IGF2BP proteins from HEK293T nuclear extract using synthetic CRD RNA fragments, CRD1 and CRD2, with (m 6 A) or without (A) m 6 A modifications. Images are representative of 3 independent experiments. ( f ) Relative firefly luciferase (Fluc) activity (i.e., protein level; left) and Fluc mRNA level (right) of wild-type (CRD-wt) or mutated (CRD-mut) CRD reporters in HEK293T cells with ectopically expressed IGF2BP1, IGF2BP2, or IGF2BP3. ( g ) RIP-qPCR detecting the in vivo binding of Flag-IGF2BPs to the transcripts of CRD-wt or CRD-mut luciferase reporter in HEK293T cells. ( h and i ) Relative luciferase activity of CRD-wt or CRD-mut in Hela cells with or without stable knockdown of IGF2BPs (h) or METTL14 (i). ( j ) Relative luciferase activity of CRD-wt or CRD-mut in METTL14 stable knockdown or control Hela cells with ectopic expression of IGF2BPs . For all luciferase assays, the Fluc/Rluc ratio (representing luciferase activity) of CRD-wt with empty vector or shNS was used for normalization. Values are mean±s.d. of n =3 independent experiments, and two-tailed Student’s t -tests were used in b , c , d , f , g , h , i , j . (**, P
    Figure Legend Snippet: IGF2BPs regulate MYC expression through binding to methylated CRD ( a ) Distribution of m 6 A peaks across MYC mRNA transcript. The coding region instability determinant (CRD) region is highlighted in yellow. m 6 A-seq was repeated twice while RIP-seq was performed once.( b ) RIP-qPCR showing the association of MYC CRD with FLAG-tagged IGF2BPs in HEK293T cells. ( c ) Enrichment of m 6 A modification in MYC CRD as detected by gene specific m 6 A qPCR assay. ( d ) RIP-qPCR showing the binding of METTL3 and METTL14 to the MYC CRD. ( e ) RNA pulldown of endogenous IGF2BP proteins from HEK293T nuclear extract using synthetic CRD RNA fragments, CRD1 and CRD2, with (m 6 A) or without (A) m 6 A modifications. Images are representative of 3 independent experiments. ( f ) Relative firefly luciferase (Fluc) activity (i.e., protein level; left) and Fluc mRNA level (right) of wild-type (CRD-wt) or mutated (CRD-mut) CRD reporters in HEK293T cells with ectopically expressed IGF2BP1, IGF2BP2, or IGF2BP3. ( g ) RIP-qPCR detecting the in vivo binding of Flag-IGF2BPs to the transcripts of CRD-wt or CRD-mut luciferase reporter in HEK293T cells. ( h and i ) Relative luciferase activity of CRD-wt or CRD-mut in Hela cells with or without stable knockdown of IGF2BPs (h) or METTL14 (i). ( j ) Relative luciferase activity of CRD-wt or CRD-mut in METTL14 stable knockdown or control Hela cells with ectopic expression of IGF2BPs . For all luciferase assays, the Fluc/Rluc ratio (representing luciferase activity) of CRD-wt with empty vector or shNS was used for normalization. Values are mean±s.d. of n =3 independent experiments, and two-tailed Student’s t -tests were used in b , c , d , f , g , h , i , j . (**, P

    Techniques Used: Expressing, Binding Assay, Methylation, Real-time Polymerase Chain Reaction, Modification, Luciferase, Activity Assay, In Vivo, Plasmid Preparation, Two Tailed Test

    Selective binding of IGF2BPs to m 6 A-modified RNAs ( a ) Identification of m 6 A specific binding proteins by RNA affinity chromatography using ssRNA probes with methylated (red) or unmethylated (green) adenosine. Silver staining (lower left) and Western blotting (lower right) showed selective pulldown of ~68kDa IGF2BP proteins from HEK293T nuclear extract. Western blot images were representative of 3 independent experiments. ( b ) Enrichment of m 6 A consensus sequence “GGAC” in the binding sites of RBPs. The three IGF2BP paralogues were shown in red, while the YTH domain proteins were shown in orange. ( c ) Quantification of m 6 A/A and m 6 A/AGCU ratios by LC-MS/MS in RNAs bound by ectopically expressed IGF2BP1 (chicken ZBP1), IGF2BP2 (human), or IGF2BP3 (human). Values are mean of n =2 independent experiments and individual data points are showed. ( d ) Overlap of IGF2BP target genes identified by RIP-seq and published PAR-CLIP in HEK293T cells. RIP-seq was performed once. P value was calculated by Fisher’s test. ( e ) Venn diagram showing the numbers of shared high-confidence targets ( i.e. , CLIP+RIP targets) amongst IGF2BP paralogues. P value was calculated by Fisher’s test. ( f ) Top consensus sequences of IGF2BP binding sites and the m 6 A motif detected by HOMER Motif analysis with PAR-CLIP data. ( g ) Pie charts showing numbers and percentages of IGF2BP high-confidence target genes that contain m 6 A peaks. The m 6 A-seq data was reported in Ref. 3 . ( h ) Metagene profiles of enrichment of IGF2BP binding sites and m 6 A modifications across mRNA transcriptome. ( i ) Percentages of various RNA species bound by IGF2BPs. ( j ) The distribution (upper) and enrichment (lower) of IGF2BPs binding peaks within different gene reions. The enrichment was determined by the proportion of IGF2BPs binding peaks normalized by the length of the region. Analyses in i and j were performed twice with similar results. ( k ) In vivo binding of Flag-IGF2BP2 to representative target genes in METTL14 knockdown or control HEK293T cells. Values are mean±s.d. of n =3 independent experiments. *, P
    Figure Legend Snippet: Selective binding of IGF2BPs to m 6 A-modified RNAs ( a ) Identification of m 6 A specific binding proteins by RNA affinity chromatography using ssRNA probes with methylated (red) or unmethylated (green) adenosine. Silver staining (lower left) and Western blotting (lower right) showed selective pulldown of ~68kDa IGF2BP proteins from HEK293T nuclear extract. Western blot images were representative of 3 independent experiments. ( b ) Enrichment of m 6 A consensus sequence “GGAC” in the binding sites of RBPs. The three IGF2BP paralogues were shown in red, while the YTH domain proteins were shown in orange. ( c ) Quantification of m 6 A/A and m 6 A/AGCU ratios by LC-MS/MS in RNAs bound by ectopically expressed IGF2BP1 (chicken ZBP1), IGF2BP2 (human), or IGF2BP3 (human). Values are mean of n =2 independent experiments and individual data points are showed. ( d ) Overlap of IGF2BP target genes identified by RIP-seq and published PAR-CLIP in HEK293T cells. RIP-seq was performed once. P value was calculated by Fisher’s test. ( e ) Venn diagram showing the numbers of shared high-confidence targets ( i.e. , CLIP+RIP targets) amongst IGF2BP paralogues. P value was calculated by Fisher’s test. ( f ) Top consensus sequences of IGF2BP binding sites and the m 6 A motif detected by HOMER Motif analysis with PAR-CLIP data. ( g ) Pie charts showing numbers and percentages of IGF2BP high-confidence target genes that contain m 6 A peaks. The m 6 A-seq data was reported in Ref. 3 . ( h ) Metagene profiles of enrichment of IGF2BP binding sites and m 6 A modifications across mRNA transcriptome. ( i ) Percentages of various RNA species bound by IGF2BPs. ( j ) The distribution (upper) and enrichment (lower) of IGF2BPs binding peaks within different gene reions. The enrichment was determined by the proportion of IGF2BPs binding peaks normalized by the length of the region. Analyses in i and j were performed twice with similar results. ( k ) In vivo binding of Flag-IGF2BP2 to representative target genes in METTL14 knockdown or control HEK293T cells. Values are mean±s.d. of n =3 independent experiments. *, P

    Techniques Used: Binding Assay, Modification, Affinity Chromatography, Methylation, Silver Staining, Western Blot, Sequencing, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Cross-linking Immunoprecipitation, In Vivo

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

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

    Journal: Cancer Cell

    doi: 10.1016/j.ccell.2018.08.017

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

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

    30) Product Images from "Transposase-assisted tagmentation of RNA/DNA hybrid duplexes"

    Article Title: Transposase-assisted tagmentation of RNA/DNA hybrid duplexes

    Journal: eLife

    doi: 10.7554/eLife.54919

    Quality assessment of TRACE-seq. ( a ) Gene expression measured by two technical replicates of TRACE-seq with 20 ng total RNA as input are shown as scatter plots. Pearson's product-moment correlations are displayed in the upper left corner. ( b ) Gene expression measured by two technical replicates of TRACE-seq with 2 ng total RNA as input are shown as scatter plots. Pearson's product-moment correlations are displayed in the upper left corner. ( c ) Scatterplots showing gene expression values for TRACE-seq and NEBNext Ultra II RNA kit with 10 ng mRNA as input (left), and for TRACE-seq with 10 ng mRNA as input and Smart-seq2 with 20 ng total RNA as input (right). All expressed genes (FPKM > 0.5) are included. Pearson's product-moment correlation is displayed in the upper left corner. ( d ) Distribution of the insert size in NEBNext Ultra II RNA library, Smart-seq2 libraries and TRACE-seq libraries with different amount of RNA as input, respectively. ( e ) Median coefficient of variation of gene coverage over the 1000 most highly expressed transcripts in NEBNext Ultra II RNA library, Smart-seq2 libraries and TRACE-seq libraries with different amount of RNA as input, respectively. ( f ) Comparison of read coverage over gene body for NEBNext Ultra II RNA kit, Smart-seq2 and TRACE-seq with different amount of RNA as input. Transcripts were grouped according to annotated lengths and analyzed separately, with the transcript length ranges indicated (top right). The read coverage over gene body is displayed along with gene body percentile from 5’ to 3’ end. ( g ) Library complexity for each library shown by plotting the number of uniquely occurring read-pairs with respect to total number of sampled read-pairs. ( h ) Assessment of RNA Integrity number (RIN). RNA of high RIN score (9.5) was used as input, which allows us to rule out the possibility that the 3’ end bias of gene body coverage is due to RNA degradation. ( i ) Nucleotide versus cycle (NVC) plots showing percentage of observed bases at each position of the first 30 bases of each sequencing read from TRACE-seq library with 10 ng mRNA and 20 ng total RNA as input. ( j ) WebLogo plot of sequence conservations of the first 10 bases of all sequencing reads from TRACE-seq library with 10 ng mRNA as input. The overall height of the stack indicates the sequence conservation at that position (measured in bits), while the height of symbols within the stack indicates the relative frequency of each nucleic acid at that position.
    Figure Legend Snippet: Quality assessment of TRACE-seq. ( a ) Gene expression measured by two technical replicates of TRACE-seq with 20 ng total RNA as input are shown as scatter plots. Pearson's product-moment correlations are displayed in the upper left corner. ( b ) Gene expression measured by two technical replicates of TRACE-seq with 2 ng total RNA as input are shown as scatter plots. Pearson's product-moment correlations are displayed in the upper left corner. ( c ) Scatterplots showing gene expression values for TRACE-seq and NEBNext Ultra II RNA kit with 10 ng mRNA as input (left), and for TRACE-seq with 10 ng mRNA as input and Smart-seq2 with 20 ng total RNA as input (right). All expressed genes (FPKM > 0.5) are included. Pearson's product-moment correlation is displayed in the upper left corner. ( d ) Distribution of the insert size in NEBNext Ultra II RNA library, Smart-seq2 libraries and TRACE-seq libraries with different amount of RNA as input, respectively. ( e ) Median coefficient of variation of gene coverage over the 1000 most highly expressed transcripts in NEBNext Ultra II RNA library, Smart-seq2 libraries and TRACE-seq libraries with different amount of RNA as input, respectively. ( f ) Comparison of read coverage over gene body for NEBNext Ultra II RNA kit, Smart-seq2 and TRACE-seq with different amount of RNA as input. Transcripts were grouped according to annotated lengths and analyzed separately, with the transcript length ranges indicated (top right). The read coverage over gene body is displayed along with gene body percentile from 5’ to 3’ end. ( g ) Library complexity for each library shown by plotting the number of uniquely occurring read-pairs with respect to total number of sampled read-pairs. ( h ) Assessment of RNA Integrity number (RIN). RNA of high RIN score (9.5) was used as input, which allows us to rule out the possibility that the 3’ end bias of gene body coverage is due to RNA degradation. ( i ) Nucleotide versus cycle (NVC) plots showing percentage of observed bases at each position of the first 30 bases of each sequencing read from TRACE-seq library with 10 ng mRNA and 20 ng total RNA as input. ( j ) WebLogo plot of sequence conservations of the first 10 bases of all sequencing reads from TRACE-seq library with 10 ng mRNA as input. The overall height of the stack indicates the sequence conservation at that position (measured in bits), while the height of symbols within the stack indicates the relative frequency of each nucleic acid at that position.

    Techniques Used: Expressing, Sequencing

    Performance of TRACE-seq in differential expression analysis. ( a ) Volcano plot showing differential expressed genes between undifferentiated and differentiated mESCs detected by NEBNext Ultra II RNA kit and TRACE-seq. Significantly up-regulated and down-regulated expressed genes (padj
    Figure Legend Snippet: Performance of TRACE-seq in differential expression analysis. ( a ) Volcano plot showing differential expressed genes between undifferentiated and differentiated mESCs detected by NEBNext Ultra II RNA kit and TRACE-seq. Significantly up-regulated and down-regulated expressed genes (padj

    Techniques Used: Expressing

    31) Product Images from "Gene autoregulation by 3’ UTR-derived bacterial small RNAs"

    Article Title: Gene autoregulation by 3’ UTR-derived bacterial small RNAs

    Journal: eLife

    doi: 10.7554/eLife.58836

    Hfq dependence of OppZ processing. ( A ) Schematic description of the analyzed OppZ variants. OppZ was produced natively from the genomic opp locus, expressed as mature sRNA from a plasmid (pOppZ) or cleaved from a plasmid-encoded precursor transcript including the 3’ end of oppF (pPrecursor). Expression of both plasmid-based oppZ variants was driven by a constitutive promoter. ( B ) V. cholerae wild-type, Δ oppZ , Δ hfq or Δ hfq Δ oppZ strains carrying oppA ::3XFLAG oppB ::3XFLAG genes and a control plasmid or the indicated OppZ expression plasmid were grown to stationary phase (OD 600 of 2.0). RNA samples were collected and OppZ processing was analyzed by Northern blot. 5S rRNA served as loading control. Full Northern blot images for the corresponding detail sections shown in Figure 2—figure supplement 1 .
    Figure Legend Snippet: Hfq dependence of OppZ processing. ( A ) Schematic description of the analyzed OppZ variants. OppZ was produced natively from the genomic opp locus, expressed as mature sRNA from a plasmid (pOppZ) or cleaved from a plasmid-encoded precursor transcript including the 3’ end of oppF (pPrecursor). Expression of both plasmid-based oppZ variants was driven by a constitutive promoter. ( B ) V. cholerae wild-type, Δ oppZ , Δ hfq or Δ hfq Δ oppZ strains carrying oppA ::3XFLAG oppB ::3XFLAG genes and a control plasmid or the indicated OppZ expression plasmid were grown to stationary phase (OD 600 of 2.0). RNA samples were collected and OppZ processing was analyzed by Northern blot. 5S rRNA served as loading control. Full Northern blot images for the corresponding detail sections shown in Figure 2—figure supplement 1 .

    Techniques Used: Produced, Plasmid Preparation, Expressing, Northern Blot

    Mutational analysis of the RNase E site in oppZ . ( A ) Predicted structure of the OppZ sRNA. The M2 mutation blocking cleavage by RNase E is indicated. ( B ) E. coli strains carrying the empty pXG10-SF plasmid or derivatives with the indicated oppZ gene in the 3’ UTR of gfp were grown to OD 600 = 1.0. RNA samples were analyzed for OppZ processing by Northern blot; 5S rRNA served as loading control. Full Northern blot images for the corresponding detail sections shown in Figure 3—figure supplement 3 .
    Figure Legend Snippet: Mutational analysis of the RNase E site in oppZ . ( A ) Predicted structure of the OppZ sRNA. The M2 mutation blocking cleavage by RNase E is indicated. ( B ) E. coli strains carrying the empty pXG10-SF plasmid or derivatives with the indicated oppZ gene in the 3’ UTR of gfp were grown to OD 600 = 1.0. RNA samples were analyzed for OppZ processing by Northern blot; 5S rRNA served as loading control. Full Northern blot images for the corresponding detail sections shown in Figure 3—figure supplement 3 .

    Techniques Used: Mutagenesis, Blocking Assay, Plasmid Preparation, Northern Blot

    Pulse expression of OppZ reduces oppBCDF transcript levels. ( A ) V. cholerae carrying pBAD1K- oppZ (pOppZ) or a control plasmid (pCtrl) were grown in biological triplicates to exponential phase (OD 600 of 0.5) and oppZ expression was induced by L-arabinose (0.2% final conc.). RNA samples were collected after 15 min and analyzed for OppZ levels by Northern blot; 5S rRNA served as loading control. ( B ) Samples from ( A ) were subjected to RNA-seq and average coverage of the opp operon is shown for one representative replicate. ( C ) V. cholerae Δ oppZ carrying pBAD1K- oppZ or a control plasmid were grown to late exponential phase (OD 600 of 1.0) and oppZ expression was induced by L-arabinose (0.2% final conc.) for 15 min. mRNA levels of oppABCDF were analyzed by qRT-PCR. Bars show mRNA levels upon OppZ induction compared to the control; error bars represent the SD of three biological replicates. ( D ) V. cholerae Δ oppZ strains carrying either pBAD1K-ctrl (pCtrl) or pBAD1K- oppZ (pOppZ) were grown to late exponential phase (OD 600 of 1.0) and treated with L-arabinose (0.2% final conc.) to induce sRNA expression. After 15 min of induction, rifampicin was added to terminate transcription. RNA samples were obtained at the indicated time points and oppB transcript levels were monitored by qRT-PCR. Error bars represent the SD of three biological replicates. Full Northern blot images for the corresponding detail sections shown in Figure 3—figure supplement 1 and raw data for transcript changes as determined by qRT-PCR.
    Figure Legend Snippet: Pulse expression of OppZ reduces oppBCDF transcript levels. ( A ) V. cholerae carrying pBAD1K- oppZ (pOppZ) or a control plasmid (pCtrl) were grown in biological triplicates to exponential phase (OD 600 of 0.5) and oppZ expression was induced by L-arabinose (0.2% final conc.). RNA samples were collected after 15 min and analyzed for OppZ levels by Northern blot; 5S rRNA served as loading control. ( B ) Samples from ( A ) were subjected to RNA-seq and average coverage of the opp operon is shown for one representative replicate. ( C ) V. cholerae Δ oppZ carrying pBAD1K- oppZ or a control plasmid were grown to late exponential phase (OD 600 of 1.0) and oppZ expression was induced by L-arabinose (0.2% final conc.) for 15 min. mRNA levels of oppABCDF were analyzed by qRT-PCR. Bars show mRNA levels upon OppZ induction compared to the control; error bars represent the SD of three biological replicates. ( D ) V. cholerae Δ oppZ strains carrying either pBAD1K-ctrl (pCtrl) or pBAD1K- oppZ (pOppZ) were grown to late exponential phase (OD 600 of 1.0) and treated with L-arabinose (0.2% final conc.) to induce sRNA expression. After 15 min of induction, rifampicin was added to terminate transcription. RNA samples were obtained at the indicated time points and oppB transcript levels were monitored by qRT-PCR. Error bars represent the SD of three biological replicates. Full Northern blot images for the corresponding detail sections shown in Figure 3—figure supplement 1 and raw data for transcript changes as determined by qRT-PCR.

    Techniques Used: Expressing, Plasmid Preparation, Northern Blot, RNA Sequencing Assay, Quantitative RT-PCR

    OppZ promotes transcription termination through Rho. ( A ) V. cholerae oppA ::3XFLAG oppB ::3XFLAG oppF ::3XFLAG strains with wild-type or mutated oppB start codon were grown to early stationary phase (OD 600 of 1.5). Cultures were divided in half and treated with either H 2 O or BCM (25 µg/ml final conc.) for 2 hr before protein and RNA samples were collected. OppA, OppB and OppF production were tested by Western blot and OppZ expression was monitored by Northern blot. RNAP and 5S rRNA served as loading controls for Western and Northern blots, respectively. ( B ) Biological triplicates of V. cholerae oppA ::3XFLAG oppB ::3XFLAG strains with wild-type or mutated oppB start codon were treated with BCM as described in ( A ). oppABCDF expression in the oppB start codon mutant compared to the wild-type control was analyzed by qRT-PCR. Error bars represent the SD of three biological replicates. ( C ) Triplicate samples from ( B ) were subjected to Term-seq and average coverage of the opp operon is shown for one representative replicate. The coverage cut-off was set at the maximum coverage of annotated genes. ( D ) V. cholerae oppA ::3XFLAG oppB ::3XFLAG strains carrying a control plasmid (pMD397) or a plasmid expressing regulator OppZ (pMD398) were treated with BCM as described in ( A ). OppA and OppB production were tested by Western blot and expression of native OppZ and regulator OppZ was monitored on Northern blot using oligonucleotides binding to the respective loop sequence variants. RNAP and 5S rRNA served as loading controls for Western and Northern blots, respectively. ( E ) Levels of oppABCDF in the experiment described in ( D ) were analyzed by qRT-PCR. Error bars represent the SD of three biological replicates. Full blot images for the corresponding detail sections shown in Figure 5 and raw data for transcript changes as determined by qRT-PCR.
    Figure Legend Snippet: OppZ promotes transcription termination through Rho. ( A ) V. cholerae oppA ::3XFLAG oppB ::3XFLAG oppF ::3XFLAG strains with wild-type or mutated oppB start codon were grown to early stationary phase (OD 600 of 1.5). Cultures were divided in half and treated with either H 2 O or BCM (25 µg/ml final conc.) for 2 hr before protein and RNA samples were collected. OppA, OppB and OppF production were tested by Western blot and OppZ expression was monitored by Northern blot. RNAP and 5S rRNA served as loading controls for Western and Northern blots, respectively. ( B ) Biological triplicates of V. cholerae oppA ::3XFLAG oppB ::3XFLAG strains with wild-type or mutated oppB start codon were treated with BCM as described in ( A ). oppABCDF expression in the oppB start codon mutant compared to the wild-type control was analyzed by qRT-PCR. Error bars represent the SD of three biological replicates. ( C ) Triplicate samples from ( B ) were subjected to Term-seq and average coverage of the opp operon is shown for one representative replicate. The coverage cut-off was set at the maximum coverage of annotated genes. ( D ) V. cholerae oppA ::3XFLAG oppB ::3XFLAG strains carrying a control plasmid (pMD397) or a plasmid expressing regulator OppZ (pMD398) were treated with BCM as described in ( A ). OppA and OppB production were tested by Western blot and expression of native OppZ and regulator OppZ was monitored on Northern blot using oligonucleotides binding to the respective loop sequence variants. RNAP and 5S rRNA served as loading controls for Western and Northern blots, respectively. ( E ) Levels of oppABCDF in the experiment described in ( D ) were analyzed by qRT-PCR. Error bars represent the SD of three biological replicates. Full blot images for the corresponding detail sections shown in Figure 5 and raw data for transcript changes as determined by qRT-PCR.

    Techniques Used: Western Blot, Expressing, Northern Blot, Mutagenesis, Quantitative RT-PCR, Plasmid Preparation, Binding Assay, Sequencing

    OppZ-dependent repression of OppA and OppB protein levels. ( A ) V. cholerae wild-type and Δ oppZ strains carrying the oppA ::3XFLAG and oppB ::3XFLAG genes and either a control plasmid or a constitutive OppZ expression plasmid were grown to obtain protein and RNA samples at the indicated OD 600 . OppA and OppB production were analyzed by Western blot and OppZ expression was tested by Northern blot. RNAP and 5S rRNA served as loading controls for Western and Northern blots, respectively. ( B ) Quantification of ( A ), bars show fold regulation of OppA and OppB in Δ oppZ compared to the wild-type; error bars represent the SD of four biological replicates. ( C ) Quantification of ( A ), bars show fold regulation of OppA and OppB upon OppZ overexpression in the Δ oppZ background compared to the wild-type control; error bars represent the SD of four biological replicates. Quantification of OppAB protein levels from Western blots and full blot images for the corresponding detail sections shown in Figure 8—figure supplement 1 .
    Figure Legend Snippet: OppZ-dependent repression of OppA and OppB protein levels. ( A ) V. cholerae wild-type and Δ oppZ strains carrying the oppA ::3XFLAG and oppB ::3XFLAG genes and either a control plasmid or a constitutive OppZ expression plasmid were grown to obtain protein and RNA samples at the indicated OD 600 . OppA and OppB production were analyzed by Western blot and OppZ expression was tested by Northern blot. RNAP and 5S rRNA served as loading controls for Western and Northern blots, respectively. ( B ) Quantification of ( A ), bars show fold regulation of OppA and OppB in Δ oppZ compared to the wild-type; error bars represent the SD of four biological replicates. ( C ) Quantification of ( A ), bars show fold regulation of OppA and OppB upon OppZ overexpression in the Δ oppZ background compared to the wild-type control; error bars represent the SD of four biological replicates. Quantification of OppAB protein levels from Western blots and full blot images for the corresponding detail sections shown in Figure 8—figure supplement 1 .

    Techniques Used: Plasmid Preparation, Expressing, Western Blot, Northern Blot, Over Expression

    Influence of OppBCDF translation on OppZ expression. ( A ) The depicted mutations were individually inserted into the opp locus to inactivate the start codons of oppB , oppC , oppD or oppF or to insert STOP codons at the positions 2, 15, 65, 115 or 215 of oppB . ( B ) V. cholerae oppA ::3XFLAG oppB ::3XFLAG strains with the described opp mutations were grown: wild-type (lane 1), the oppB start codon mutated (lane 2), a STOP codon inserted at the 2 nd , 15 th , 65 th , 115 th or 215 th codon of oppB (lanes 3–7) or mutated start codons of oppC , oppD or oppF (lanes 8–10). At stationary phase (OD 600 of 2.0), protein and RNA samples were collected and tested for OppA and OppB production by Western blot and for OppZ expression by Northern blot. RNAP and 5S rRNA served as loading controls for Western and Northern blots, respectively. Full Northern and Western blot images for the corresponding detail sections shown in Figure 6 .
    Figure Legend Snippet: Influence of OppBCDF translation on OppZ expression. ( A ) The depicted mutations were individually inserted into the opp locus to inactivate the start codons of oppB , oppC , oppD or oppF or to insert STOP codons at the positions 2, 15, 65, 115 or 215 of oppB . ( B ) V. cholerae oppA ::3XFLAG oppB ::3XFLAG strains with the described opp mutations were grown: wild-type (lane 1), the oppB start codon mutated (lane 2), a STOP codon inserted at the 2 nd , 15 th , 65 th , 115 th or 215 th codon of oppB (lanes 3–7) or mutated start codons of oppC , oppD or oppF (lanes 8–10). At stationary phase (OD 600 of 2.0), protein and RNA samples were collected and tested for OppA and OppB production by Western blot and for OppZ expression by Northern blot. RNAP and 5S rRNA served as loading controls for Western and Northern blots, respectively. Full Northern and Western blot images for the corresponding detail sections shown in Figure 6 .

    Techniques Used: Expressing, Western Blot, Northern Blot

    Translational control of OppZ synthesis. ( A ) V. cholerae wild-type and oppB ATC strains were grown to stationary phase (OD 600 of 2.0) and treated with rifampicin to terminate transcription. RNA samples were obtained at the indicated time points and OppZ transcript levels were monitored by Northern blot and normalized to 5S rRNA levels as loading control. Error bars represent the SD of three biological replicates. ( B ) V. cholerae wild-type and oppB ATC strains carrying either a control plasmid (pCtrl) or an inducible oppB complementation plasmid (pOppB) were grown to late exponential phase (OD 600 of 1.0) and oppB expression was induced by the addition of L-arabinose (0.2% final conc.). Protein and RNA samples were obtained after 60 min and tested for OppA and OppB production by Western blot and for OppZ expression by Northern blot. RNAP and 5S rRNA served as loading controls for Western and Northern blots, respectively. Quantification of OppZ levels in wild-type and oppB ATC cells from Northern blots and full blot images for the corresponding detail sections shown in Figure 4—figure supplement 1 .
    Figure Legend Snippet: Translational control of OppZ synthesis. ( A ) V. cholerae wild-type and oppB ATC strains were grown to stationary phase (OD 600 of 2.0) and treated with rifampicin to terminate transcription. RNA samples were obtained at the indicated time points and OppZ transcript levels were monitored by Northern blot and normalized to 5S rRNA levels as loading control. Error bars represent the SD of three biological replicates. ( B ) V. cholerae wild-type and oppB ATC strains carrying either a control plasmid (pCtrl) or an inducible oppB complementation plasmid (pOppB) were grown to late exponential phase (OD 600 of 1.0) and oppB expression was induced by the addition of L-arabinose (0.2% final conc.). Protein and RNA samples were obtained after 60 min and tested for OppA and OppB production by Western blot and for OppZ expression by Northern blot. RNAP and 5S rRNA served as loading controls for Western and Northern blots, respectively. Quantification of OppZ levels in wild-type and oppB ATC cells from Northern blots and full blot images for the corresponding detail sections shown in Figure 4—figure supplement 1 .

    Techniques Used: Northern Blot, Plasmid Preparation, Expressing, Western Blot

    Hfq-dependent, post-transcriptional repression of OppBCDF by OppZ. ( A ) E. coli Δ hfq strains carrying the translational oppB-gfp reporter plasmid and either a control plasmid or the indicated OppZ expression plasmids were grown to OD 600 = 1.0 and fluorophore production was measured. GFP levels of the control strain were set to 1. Error bars represent the SD of three biological replicates. ( B ) E. coli wild-type or Δ hfq strains carrying the translational oppB-gfp reporter plasmid and either a control plasmid or the indicated OppZ expression plasmids were grown to OD 600 = 1.0. RNA samples were analyzed for OppZ levels by Northern blot; 5S rRNA served as loading control. ( C ) E. coli strains carrying translational reporter plasmids with the indicated parts of the opp operon placed between mKate2 and gfp were co-transformed with a control plasmid or the respective OppZ expression plasmids. Transcription of the reporter and oppZ were driven by constitutive promoters. Cells were grown to OD 600 = 1.0 and fluorophore production was measured. mKate and GFP levels of strains carrying the control plasmid were set to 1. Error bars represent the SD of three biological replicates. Full Northern blot images for the corresponding detail sections shown in Figure 3—figure supplement 2 and raw data for fluorescence measurements.
    Figure Legend Snippet: Hfq-dependent, post-transcriptional repression of OppBCDF by OppZ. ( A ) E. coli Δ hfq strains carrying the translational oppB-gfp reporter plasmid and either a control plasmid or the indicated OppZ expression plasmids were grown to OD 600 = 1.0 and fluorophore production was measured. GFP levels of the control strain were set to 1. Error bars represent the SD of three biological replicates. ( B ) E. coli wild-type or Δ hfq strains carrying the translational oppB-gfp reporter plasmid and either a control plasmid or the indicated OppZ expression plasmids were grown to OD 600 = 1.0. RNA samples were analyzed for OppZ levels by Northern blot; 5S rRNA served as loading control. ( C ) E. coli strains carrying translational reporter plasmids with the indicated parts of the opp operon placed between mKate2 and gfp were co-transformed with a control plasmid or the respective OppZ expression plasmids. Transcription of the reporter and oppZ were driven by constitutive promoters. Cells were grown to OD 600 = 1.0 and fluorophore production was measured. mKate and GFP levels of strains carrying the control plasmid were set to 1. Error bars represent the SD of three biological replicates. Full Northern blot images for the corresponding detail sections shown in Figure 3—figure supplement 2 and raw data for fluorescence measurements.

    Techniques Used: Plasmid Preparation, Expressing, Northern Blot, Transformation Assay, Fluorescence

    Hfq dependence of OppZ stability. V. cholerae wild-type and Δ hfq strains were grown to early stationary phase (OD 600 of 1.5) and treated with rifampicin to terminate transcription. RNA samples were obtained at the indicated time points and OppZ transcript levels were monitored by Northern blot and normalized to 5S rRNA levels as loading control. Error bars represent the SD of three biological replicates. Quantification of OppZ levels in wild-type and Δhfq cells from Northern blots.
    Figure Legend Snippet: Hfq dependence of OppZ stability. V. cholerae wild-type and Δ hfq strains were grown to early stationary phase (OD 600 of 1.5) and treated with rifampicin to terminate transcription. RNA samples were obtained at the indicated time points and OppZ transcript levels were monitored by Northern blot and normalized to 5S rRNA levels as loading control. Error bars represent the SD of three biological replicates. Quantification of OppZ levels in wild-type and Δhfq cells from Northern blots.

    Techniques Used: Northern Blot

    32) Product Images from "In Vivo Study of Key Transcription Factors in Muscle Satellite Cells by CRISPR/Cas9/AAV9-sgRNA Mediated Genome Editing"

    Article Title: In Vivo Study of Key Transcription Factors in Muscle Satellite Cells by CRISPR/Cas9/AAV9-sgRNA Mediated Genome Editing

    Journal: bioRxiv

    doi: 10.1101/797746

    CRISPR/Cas9/AAV9 mediated genome editing of Bcl6 and Pknox2 led to distinct effects on SC activation and muscle regeneration. ( A ) Design and selection of sgRNAs targeting Bcl6 locus. SgRNA2 and sgRNA3 were selected to delete a 133 bp of exon 3. ( B ) Schematic illustration of the experimental design for in vivo genome editing of Bcl6 locus followed by analysis of its effect on SCs and muscle regeneration. Pax7 Cas9 mice were administrated with high dose of control or AAV9-dual sgBcl6 virus at P10 and SCs were isolated four weeks later for analyses. To assay for muscle regeneration, BaCl 2 was intramuscularly injected at the TA region of one leg at least six weeks after AAV9 virus administration. The injected muscle was harvested 3.5 or 5 days post injury and subject to analysis. ( C ) Genomic DNAs were isolated from the above sorted SCs and PCR analysis was performed to test the cleavage efficiency. Wide type (568 bp) and cleaved fragments (∼435 bp) are shown by arrowheads. ( D ) The number of SCs isolated from the above control or dual sgBcl6 virus treated mice (n = 6 in each group). M: million. ( E ) The above FISCs were labeled with EdU for 24 hrs and the percentage of EdU positive cells was quantified (n = 3 in each group). Scale bar, 50 μm. ( F ) RNAs were isolated from the above SCs cultured for 24 hrs and subject to RNA-seq analysis. Differentially expressed genes were identified with blue and red dots representing down- or up-regulated genes, respectively. ( G ) GO analysis of the above up-regulated genes was performed and the top five enriched items are shown in y axis. X axis shows the −log 10 [ P value] of the GO terms. ( H ) Genomic snapshots showing two examples of significantly up-regulated genes in sgBcl6 vs. ctrl treatment. ( I ) Immunostaining of Pax7 (red) and Laminin (green) were performed on the uninjured contralateral TA muscle (Left). The number of Pax7 positive cells per 100 fibers was counted (Right) (n = 3 in each group). Scale bar, 50 μm. ( J ) Average fiber size of the above TA muscle was quantified (n = 3 in each group). ( K ) H E staining was performed 3.5 or 5 days post BaCl 2 injection. Uninjured TA muscle was used as control (Left). Scale bar, 100 μm. ( L ) Immunostaining of eMyHC (red) and Laminin (green) on the above TA muscle sections (Left) and the number of eMyHC+ myofibers was counted (Right) (n = 4 in each group). Scale bar, 50 μm. ( M ) Design and selection of sgRNAs targeting Pknox2 locus. SgRNA3 and sgRNA4 were selected to delete a 98 bp of exon 6. ( N ) Schematic illustration of the experimental design for in vivo genome editing of Pknox2 locus and analysis of its effect on SCs. Pax7 Cas9 mice were administrated with high dose of control or AAV9-dual sgPknox2 virus at P10 and SCs were isolated four weeks later for analysis. ( O ) Genomic DNAs were isolated from the above sorted SCs and PCR analysis was performed to test the cleavage efficiency. Wide type (578 bp) and cleaved fragments (∼480 bp) are shown by arrowheads. ( P ) The number of SCs isolated from the above control or dual sgPknox2 virus treated mice (n = 3 in each group). M: million. ( Q ) The above FISCs were labeled with EdU for 24 hrs and the percentage of EdU positive cells was quantified (n = 3 in each group). Scale bar, 50 μm. All the bar graphs are presented as mean ± s.d. *P
    Figure Legend Snippet: CRISPR/Cas9/AAV9 mediated genome editing of Bcl6 and Pknox2 led to distinct effects on SC activation and muscle regeneration. ( A ) Design and selection of sgRNAs targeting Bcl6 locus. SgRNA2 and sgRNA3 were selected to delete a 133 bp of exon 3. ( B ) Schematic illustration of the experimental design for in vivo genome editing of Bcl6 locus followed by analysis of its effect on SCs and muscle regeneration. Pax7 Cas9 mice were administrated with high dose of control or AAV9-dual sgBcl6 virus at P10 and SCs were isolated four weeks later for analyses. To assay for muscle regeneration, BaCl 2 was intramuscularly injected at the TA region of one leg at least six weeks after AAV9 virus administration. The injected muscle was harvested 3.5 or 5 days post injury and subject to analysis. ( C ) Genomic DNAs were isolated from the above sorted SCs and PCR analysis was performed to test the cleavage efficiency. Wide type (568 bp) and cleaved fragments (∼435 bp) are shown by arrowheads. ( D ) The number of SCs isolated from the above control or dual sgBcl6 virus treated mice (n = 6 in each group). M: million. ( E ) The above FISCs were labeled with EdU for 24 hrs and the percentage of EdU positive cells was quantified (n = 3 in each group). Scale bar, 50 μm. ( F ) RNAs were isolated from the above SCs cultured for 24 hrs and subject to RNA-seq analysis. Differentially expressed genes were identified with blue and red dots representing down- or up-regulated genes, respectively. ( G ) GO analysis of the above up-regulated genes was performed and the top five enriched items are shown in y axis. X axis shows the −log 10 [ P value] of the GO terms. ( H ) Genomic snapshots showing two examples of significantly up-regulated genes in sgBcl6 vs. ctrl treatment. ( I ) Immunostaining of Pax7 (red) and Laminin (green) were performed on the uninjured contralateral TA muscle (Left). The number of Pax7 positive cells per 100 fibers was counted (Right) (n = 3 in each group). Scale bar, 50 μm. ( J ) Average fiber size of the above TA muscle was quantified (n = 3 in each group). ( K ) H E staining was performed 3.5 or 5 days post BaCl 2 injection. Uninjured TA muscle was used as control (Left). Scale bar, 100 μm. ( L ) Immunostaining of eMyHC (red) and Laminin (green) on the above TA muscle sections (Left) and the number of eMyHC+ myofibers was counted (Right) (n = 4 in each group). Scale bar, 50 μm. ( M ) Design and selection of sgRNAs targeting Pknox2 locus. SgRNA3 and sgRNA4 were selected to delete a 98 bp of exon 6. ( N ) Schematic illustration of the experimental design for in vivo genome editing of Pknox2 locus and analysis of its effect on SCs. Pax7 Cas9 mice were administrated with high dose of control or AAV9-dual sgPknox2 virus at P10 and SCs were isolated four weeks later for analysis. ( O ) Genomic DNAs were isolated from the above sorted SCs and PCR analysis was performed to test the cleavage efficiency. Wide type (578 bp) and cleaved fragments (∼480 bp) are shown by arrowheads. ( P ) The number of SCs isolated from the above control or dual sgPknox2 virus treated mice (n = 3 in each group). M: million. ( Q ) The above FISCs were labeled with EdU for 24 hrs and the percentage of EdU positive cells was quantified (n = 3 in each group). Scale bar, 50 μm. All the bar graphs are presented as mean ± s.d. *P

    Techniques Used: CRISPR, Activation Assay, Selection, In Vivo, Mouse Assay, Isolation, Injection, Polymerase Chain Reaction, Labeling, Cell Culture, RNA Sequencing Assay, Immunostaining, Staining

    Attempts of CRISPR/Cas9/AAV9 mediated genome editing in quiescent SCs. ( A ) Schematic illustration to show the generation of the inducible muscle-specific Cas9 knockin mouse (Pax7 ER-Cas9 ). ( B ) Schematic illustration of the first strategy to edit quiescent SCs (QSCs) in the above generated mouse. High dose of AAV9-sgMyoD or control virus was injected through IM to four weeks old Pax7 ER-Cas9 mice. After two weeks, Tamoxifen (Tmx) was injected intraperitoneally (IP) for five consecutive days to induce Cas9 and GFP expression. The mice were sacrificed for SC isolation and analysis after another three weeks. ( C ) FACS plot showing efficient induction of Cas9-GFP expression in SCs from the above mice. The gate P4 indicates the population of Cas9-GFP positive SCs isolated for following analysis. ( D ) Genomic DNAs (gDNA) were isolated from the above sorted SCs and PCR was performed to amplify the DsRed or Cas9 coding region. Genomic DNA from Pax7 ER-Cas9 mice without AAV9 administration was used as negative control and the AAV9-MyoD-sgRNA2 plasmid was used as positive control. ( E ) Surveyor assay was performed to detect the editing efficiency at MyoD locus in the above FISCs. A 660 bp amplicon of wild type MyoD locus is shown. ( F ) Relative expression of MyoD , Myogenin and MyHC mRNAs were detected in the above SCs cultured for four days (n = 3 in each group). (G) Schematic illustration of the second strategy to edit QSCs in Pax7 ER-Cas9 mice. High dose of AAV9-sgMyoD or control virus was injected through IM at P10. Four weeks later, Tmx was injected via IP for five consecutive days and the mice were sacrificed for SC isolation and analysis after another three weeks. (H) FACS plot showing the gating strategy to sort out Cas9-GFP positive SCs from the above treated mice (Upper panel). A stronger DsRed signal compared to Strategy 1 (Lower panel) indicates a higher transduction efficiency. ( I ) Transduction of AAV9 virus in SCs was examined as describe in (D). ( J ) Editing efficiency was examined by Surveyor assay as in (E). ( K ) SCs from the above treated mice were cultured for two days and IF stained for MyoD (Left) and the number of positively stained cells was counted (Right) (n=2 in each group). Scale bar, 50µm. ( L ) Schematic illustration of the third strategy to edit QSCs from adult mice. A middle dose of AAV9-dual sgMyoD or control virus was injected through IP into Pax7 ER-Cas9 mice at P2 and another high dose of the same virus was administrated at P10 through IM. Four weeks later, Tmx was administrated via IP for five consecutive days and the mice were sacrificed for SC isolation and analysis after another three weeks. ( M ) Transduction of AAV9 virus in SCs was examined as describe in (D). ( N - O ) Editing efficiency was examined by PCR analysis targeting MyoD locus ( N ) or Surveyor assay( O ) as in (E). ( P ) SCs from the above treated mice were cultured for two days and IF stained for MyoD (Left) and the number of positively stained cells was counted (Right) (n = 3 in each group). Scale bar, 50µm. ( Q ) Relative expressions of MyoD , Myogenin and MyHC mRNAs were detected in the above SCs cultured for four days (n = 3 in each group). All qRT-PCR data were normalized to 18S or GAPDH mRNA. All the bar graphs are presented as mean ± s.d. ns, no significance.
    Figure Legend Snippet: Attempts of CRISPR/Cas9/AAV9 mediated genome editing in quiescent SCs. ( A ) Schematic illustration to show the generation of the inducible muscle-specific Cas9 knockin mouse (Pax7 ER-Cas9 ). ( B ) Schematic illustration of the first strategy to edit quiescent SCs (QSCs) in the above generated mouse. High dose of AAV9-sgMyoD or control virus was injected through IM to four weeks old Pax7 ER-Cas9 mice. After two weeks, Tamoxifen (Tmx) was injected intraperitoneally (IP) for five consecutive days to induce Cas9 and GFP expression. The mice were sacrificed for SC isolation and analysis after another three weeks. ( C ) FACS plot showing efficient induction of Cas9-GFP expression in SCs from the above mice. The gate P4 indicates the population of Cas9-GFP positive SCs isolated for following analysis. ( D ) Genomic DNAs (gDNA) were isolated from the above sorted SCs and PCR was performed to amplify the DsRed or Cas9 coding region. Genomic DNA from Pax7 ER-Cas9 mice without AAV9 administration was used as negative control and the AAV9-MyoD-sgRNA2 plasmid was used as positive control. ( E ) Surveyor assay was performed to detect the editing efficiency at MyoD locus in the above FISCs. A 660 bp amplicon of wild type MyoD locus is shown. ( F ) Relative expression of MyoD , Myogenin and MyHC mRNAs were detected in the above SCs cultured for four days (n = 3 in each group). (G) Schematic illustration of the second strategy to edit QSCs in Pax7 ER-Cas9 mice. High dose of AAV9-sgMyoD or control virus was injected through IM at P10. Four weeks later, Tmx was injected via IP for five consecutive days and the mice were sacrificed for SC isolation and analysis after another three weeks. (H) FACS plot showing the gating strategy to sort out Cas9-GFP positive SCs from the above treated mice (Upper panel). A stronger DsRed signal compared to Strategy 1 (Lower panel) indicates a higher transduction efficiency. ( I ) Transduction of AAV9 virus in SCs was examined as describe in (D). ( J ) Editing efficiency was examined by Surveyor assay as in (E). ( K ) SCs from the above treated mice were cultured for two days and IF stained for MyoD (Left) and the number of positively stained cells was counted (Right) (n=2 in each group). Scale bar, 50µm. ( L ) Schematic illustration of the third strategy to edit QSCs from adult mice. A middle dose of AAV9-dual sgMyoD or control virus was injected through IP into Pax7 ER-Cas9 mice at P2 and another high dose of the same virus was administrated at P10 through IM. Four weeks later, Tmx was administrated via IP for five consecutive days and the mice were sacrificed for SC isolation and analysis after another three weeks. ( M ) Transduction of AAV9 virus in SCs was examined as describe in (D). ( N - O ) Editing efficiency was examined by PCR analysis targeting MyoD locus ( N ) or Surveyor assay( O ) as in (E). ( P ) SCs from the above treated mice were cultured for two days and IF stained for MyoD (Left) and the number of positively stained cells was counted (Right) (n = 3 in each group). Scale bar, 50µm. ( Q ) Relative expressions of MyoD , Myogenin and MyHC mRNAs were detected in the above SCs cultured for four days (n = 3 in each group). All qRT-PCR data were normalized to 18S or GAPDH mRNA. All the bar graphs are presented as mean ± s.d. ns, no significance.

    Techniques Used: CRISPR, Knock-In, Generated, Injection, Mouse Assay, Expressing, Isolation, FACS, Polymerase Chain Reaction, Negative Control, Plasmid Preparation, Positive Control, Amplification, Cell Culture, Transduction, Staining, Quantitative RT-PCR

    AAV9-sgRNA mediated editing of MyoD in SCs of Pax7 Cas9 mice. ( A ) Design of sgRNAs for targeting the first exon of MyoD locus. Selected sgRNA2 was marked as red and its targeted genomic sequence is shown. PAM: protospacer-adjacent motif. ( B ) Top: schematic illustration of the sgRNA-Cas9 expressing vector; Bottom: illustration of in vitro sgRNA selection assay. SgRNA-Cas9 expressing plasmid was transfected into C2C12 myoblasts and positively transfected cells were sorted out by GFP expression one day after transfection and cultured for another two days before Surveyor nuclease assay. ( C ) Agarose gel image to show the result of Surveyor nuclease assay on MyoD locus. The percentage of indel formation is shown. SgRNA2 with the highest frequency of indel formation is marked as red. ( D ) Schematic illustration of the pAAV9-sgRNA vector for in vivo sgRNA expression. ( E ) Schematic illustration of the experimental design for AAV administration and SC isolation and analysis. ( F ) Genomic DNAs from SCs of the Pax7 Cas9 mice administrated with AAV9-sgMyoD virus were subject to Surveyor assay to test the frequency of indel formation at MyoD locus. Wide type (660 bp) and cleaved bands (∼467 bp/193 bp) by Surveyor are shown by arrowheads. The percentage of indel formation is shown (n=2 in each group). ( G ) Three doses of AAV virus were administrated and Surveyor assay was performed as above to estimate the indel formation at MyoD locus in genomic DNAs from FISCs. Wide type (660 bp) and cleaved bands (∼467 bp/193 bp) by Surveyor are shown by arrowheads. ( H ) Quantification of the editing efficiency (Left) and frameshift mutations (Right) determined by deep sequencing of the genomic DNAs from the above (G). The percentage was calculated as the ratio of the edited reads (Left) or frameshift mutations (Right) to the total reads and presented as mean ± s.d (n=3 in each group). ( I ) The five most frequently detected indel classes under each dose of AAV9-sgMyoD virus are shown. The percentage is calculated as the ratio of reads for each indel class to the total edited reads. ( J ) FISCs from the three doses of AAV9-sgMyoD administration were cultured for two days and the MyoD protein level was examined by Western blot. α-Tubulin was used as loading control. ( K ) The number of SCs purified from Pax7 Cas9 mice administrated with different doses of AAV9-sgMyoD virus is presented as mean ± s.d (n = 3 in each group). M: million. ( L ) The above isolated SCs were cultured for two or four days and the relative expressions of Myogenin or MyHC was detected by qRT-PCR (n = 3 in each group). All qRT-PCR data were normalized to 18S mRNA and presented as mean ± s.d. *P
    Figure Legend Snippet: AAV9-sgRNA mediated editing of MyoD in SCs of Pax7 Cas9 mice. ( A ) Design of sgRNAs for targeting the first exon of MyoD locus. Selected sgRNA2 was marked as red and its targeted genomic sequence is shown. PAM: protospacer-adjacent motif. ( B ) Top: schematic illustration of the sgRNA-Cas9 expressing vector; Bottom: illustration of in vitro sgRNA selection assay. SgRNA-Cas9 expressing plasmid was transfected into C2C12 myoblasts and positively transfected cells were sorted out by GFP expression one day after transfection and cultured for another two days before Surveyor nuclease assay. ( C ) Agarose gel image to show the result of Surveyor nuclease assay on MyoD locus. The percentage of indel formation is shown. SgRNA2 with the highest frequency of indel formation is marked as red. ( D ) Schematic illustration of the pAAV9-sgRNA vector for in vivo sgRNA expression. ( E ) Schematic illustration of the experimental design for AAV administration and SC isolation and analysis. ( F ) Genomic DNAs from SCs of the Pax7 Cas9 mice administrated with AAV9-sgMyoD virus were subject to Surveyor assay to test the frequency of indel formation at MyoD locus. Wide type (660 bp) and cleaved bands (∼467 bp/193 bp) by Surveyor are shown by arrowheads. The percentage of indel formation is shown (n=2 in each group). ( G ) Three doses of AAV virus were administrated and Surveyor assay was performed as above to estimate the indel formation at MyoD locus in genomic DNAs from FISCs. Wide type (660 bp) and cleaved bands (∼467 bp/193 bp) by Surveyor are shown by arrowheads. ( H ) Quantification of the editing efficiency (Left) and frameshift mutations (Right) determined by deep sequencing of the genomic DNAs from the above (G). The percentage was calculated as the ratio of the edited reads (Left) or frameshift mutations (Right) to the total reads and presented as mean ± s.d (n=3 in each group). ( I ) The five most frequently detected indel classes under each dose of AAV9-sgMyoD virus are shown. The percentage is calculated as the ratio of reads for each indel class to the total edited reads. ( J ) FISCs from the three doses of AAV9-sgMyoD administration were cultured for two days and the MyoD protein level was examined by Western blot. α-Tubulin was used as loading control. ( K ) The number of SCs purified from Pax7 Cas9 mice administrated with different doses of AAV9-sgMyoD virus is presented as mean ± s.d (n = 3 in each group). M: million. ( L ) The above isolated SCs were cultured for two or four days and the relative expressions of Myogenin or MyHC was detected by qRT-PCR (n = 3 in each group). All qRT-PCR data were normalized to 18S mRNA and presented as mean ± s.d. *P

    Techniques Used: Mouse Assay, Sequencing, Expressing, Plasmid Preparation, In Vitro, Selection, Transfection, Cell Culture, Nuclease Assay, Agarose Gel Electrophoresis, In Vivo, Isolation, Western Blot, Purification, Quantitative RT-PCR

    Development of an AAV9-dual sgRNA strategy for precise genome editing in SCs. ( A ) Top: schematic illustration of the pAAV9-sgRNA vector used for dual sgRNAs expression; Middle: design and selection of sgRNAs targeting the first coding exon of MyoD . SgRNA2 and sgRNAc selected for in vivo use were marked as red. Bottom: the targeted genomic sequence for sgRNAc. PAM: protospacer-adjacent motif. ( B ) Schematic illustration of the experimental design for AAV-dual sgMyoD virus administration and SC isolation/analysis. The administration of middle dose of AAV9-sgRNA backbone (without sgRNA insertion) virus or single AAV9-sgMyoD virus was used as control or comparison. ( C ) PCR analysis to test the cleavage efficiency at MyoD locus in FISCs from the above injected mice. DNA from C2C12 cells co-transfected with Cas9-expressing plasmid (pX458) and AAV9-dual sgMyoD vector was used as positive control. Wide type (660 bp) and cleaved bands (∼362 bp) are shown by arrowheads. ( D ) SCs were cultured for two days and IF stained for MyoD (Left). The number of positively stained cells was counted (Right) (n = 3 in each group). Scale bar, 50 µm. ( E ) The number of FISCs sorted from the above mice (n = 3 in each group). M: million. ( F ) Relative expression of MyoD and Myogenin in the above SCs cultured for two days were determined by qRT-PCR. The qRT-PCR data were normalized to 18S mRNA and presented as mean ±s.d (n = 3 in each group). ( G ) Schematic illustration to show non-homologous end joining (NHEJ) repair of double-strand breaks (DSBs) induced by AAV9-dual sgMyoD. The edited products were divided into four groups based on whether the indels formed at either sgRNA or both target sites. Group I: editing at sgRNA2 site only; II: sgRNAc site only; III: individually at sgRNA2 and sgRNAc site; IV: simultaneously at both sites to cause deletion. ( H ) Genomic DNAs were isolated from FISCs and subject to deep sequencing to assess the editing events. The percentage of editing was calculated as the ratio of the edited reads to the total reads (n = 3 in each group). ( I ) Left: distribution of the total sequencing reads in the dual sgRNAs group. Right: distribution of the above Group IV reads. Accurate: reads with precise ligation of the two predicted cutting sites. Insertion (or deletion): reads with insertion (or deletion) at the junction site. Others: reads that do not belong to the above. The percentage was calculated as the ratio of each part to the total reads in group IV (n = 3 in each group). All the bar graphs are presented as mean ± s.d. *P
    Figure Legend Snippet: Development of an AAV9-dual sgRNA strategy for precise genome editing in SCs. ( A ) Top: schematic illustration of the pAAV9-sgRNA vector used for dual sgRNAs expression; Middle: design and selection of sgRNAs targeting the first coding exon of MyoD . SgRNA2 and sgRNAc selected for in vivo use were marked as red. Bottom: the targeted genomic sequence for sgRNAc. PAM: protospacer-adjacent motif. ( B ) Schematic illustration of the experimental design for AAV-dual sgMyoD virus administration and SC isolation/analysis. The administration of middle dose of AAV9-sgRNA backbone (without sgRNA insertion) virus or single AAV9-sgMyoD virus was used as control or comparison. ( C ) PCR analysis to test the cleavage efficiency at MyoD locus in FISCs from the above injected mice. DNA from C2C12 cells co-transfected with Cas9-expressing plasmid (pX458) and AAV9-dual sgMyoD vector was used as positive control. Wide type (660 bp) and cleaved bands (∼362 bp) are shown by arrowheads. ( D ) SCs were cultured for two days and IF stained for MyoD (Left). The number of positively stained cells was counted (Right) (n = 3 in each group). Scale bar, 50 µm. ( E ) The number of FISCs sorted from the above mice (n = 3 in each group). M: million. ( F ) Relative expression of MyoD and Myogenin in the above SCs cultured for two days were determined by qRT-PCR. The qRT-PCR data were normalized to 18S mRNA and presented as mean ±s.d (n = 3 in each group). ( G ) Schematic illustration to show non-homologous end joining (NHEJ) repair of double-strand breaks (DSBs) induced by AAV9-dual sgMyoD. The edited products were divided into four groups based on whether the indels formed at either sgRNA or both target sites. Group I: editing at sgRNA2 site only; II: sgRNAc site only; III: individually at sgRNA2 and sgRNAc site; IV: simultaneously at both sites to cause deletion. ( H ) Genomic DNAs were isolated from FISCs and subject to deep sequencing to assess the editing events. The percentage of editing was calculated as the ratio of the edited reads to the total reads (n = 3 in each group). ( I ) Left: distribution of the total sequencing reads in the dual sgRNAs group. Right: distribution of the above Group IV reads. Accurate: reads with precise ligation of the two predicted cutting sites. Insertion (or deletion): reads with insertion (or deletion) at the junction site. Others: reads that do not belong to the above. The percentage was calculated as the ratio of each part to the total reads in group IV (n = 3 in each group). All the bar graphs are presented as mean ± s.d. *P

    Techniques Used: Plasmid Preparation, Expressing, Selection, In Vivo, Sequencing, Isolation, Polymerase Chain Reaction, Injection, Mouse Assay, Transfection, Positive Control, Cell Culture, Staining, Quantitative RT-PCR, Non-Homologous End Joining, Ligation

    CRISPR/Cas9/AAV9 mediated genome editing of Myc hindered SC activation and muscle regeneration. ( A ) Design and selection of sgRNAs targeting Myc locus. SgRNA1 and sgRNAb were selected to delete a 235 bp of exon 2. ( B ) Schematic illustration of the experimental design for in vivo genome editing of Myc locus and analysis of its effect on SCs and muscle regeneration. Pax7 Cas9 mice were administrated with high dose of control or AAV9-dual sgMyc virus at P10 and SCs were isolated four weeks later for analysis. To assay for muscle regeneration, BaCl 2 was intramuscularly injected at the TA region of one leg at least six weeks after AAV9 virus administration. The injected muscle was harvested five days post injury and subject to analysis. ( C ) Genomic DNAs were isolated from the above sorted SCs and PCR analysis was performed to test the cleavage efficiency. Wide type (583 bp) and cleaved fragments (∼348 bp) are shown by arrowheads. ( D ) The above SCs were cultured for 24 hrs and relative expression of Myc mRNAs was detected by qRT-PCR. The qRT-PCR data were normalized to 18S mRNA (n=3 in each group). ( E ) Myc protein level in the above SCs was examined by Western blot. GAPDH was used as loading control. ( F ) The number of SCs isolated from the above control or dual sgMyc virus treated mice (n = 6 in each group). M: million. ( G ) The above FISCs were labeled with EdU for 24 hrs and the percentage of EdU positive cells was quantified (n = 3 in each group). Scale bar, 50 μm. ( H ) The above FISCs were IF stained for MyoD expression four hrs after seeding (Left) and the percentage of positively stained cells was quantified (Right) (n = 3 in each group). Scale bar, 50 μm. ( I ) RNAs were isolated from the above SCs cultured for 24 hrs and subject to RNA-seq analysis. Differentially expressed genes were identified with blue and red dots representing down- or up-regulated genes, respectively. ( J - K ) GO analysis of the above up- or down-regulated genes was performed and the top five enriched items are shown in y axis. X axis shows the −log 10 [ P value] of the GO terms. ( L ) Genomic snapshots showing two examples of significantly down-regulated genes in sgMyc vs. ctrl treatment. ( M ) Immunostaining of Pax7 (red) and Laminin (green) waere performed on the uninjured contralateral TA muscle (Left). The number of Pax7 positive cells per 100 fibers was counted (Right) (n = 3 in each group). Scale bar, 50 μm. ( N ) Average fiber size of the above TA muscle was quantified (n = 3 in each group). ( O ) H E staining was performed 5 days post the BaCl 2 injection. Uninjured TA muscle was used as control (Left). The regenerating myofibers with CLN per field was quantified (Right) (n = 3 in each group). Scale bar, 100 μm. ( P ) Immunostaining of eMyHC (red) and Laminin (green) on the above TA muscle sections (Left) and the number of eMyHC+ myofibers was counted (Right) (n = 3 in each group). Scale bar, 50 μm. ( Q ) The cross-sectional area (CSA) of the newly formed fibers with CLN was quantified (n = 3 in each group). All the bar graphs are presented as mean ± s.d. *P
    Figure Legend Snippet: CRISPR/Cas9/AAV9 mediated genome editing of Myc hindered SC activation and muscle regeneration. ( A ) Design and selection of sgRNAs targeting Myc locus. SgRNA1 and sgRNAb were selected to delete a 235 bp of exon 2. ( B ) Schematic illustration of the experimental design for in vivo genome editing of Myc locus and analysis of its effect on SCs and muscle regeneration. Pax7 Cas9 mice were administrated with high dose of control or AAV9-dual sgMyc virus at P10 and SCs were isolated four weeks later for analysis. To assay for muscle regeneration, BaCl 2 was intramuscularly injected at the TA region of one leg at least six weeks after AAV9 virus administration. The injected muscle was harvested five days post injury and subject to analysis. ( C ) Genomic DNAs were isolated from the above sorted SCs and PCR analysis was performed to test the cleavage efficiency. Wide type (583 bp) and cleaved fragments (∼348 bp) are shown by arrowheads. ( D ) The above SCs were cultured for 24 hrs and relative expression of Myc mRNAs was detected by qRT-PCR. The qRT-PCR data were normalized to 18S mRNA (n=3 in each group). ( E ) Myc protein level in the above SCs was examined by Western blot. GAPDH was used as loading control. ( F ) The number of SCs isolated from the above control or dual sgMyc virus treated mice (n = 6 in each group). M: million. ( G ) The above FISCs were labeled with EdU for 24 hrs and the percentage of EdU positive cells was quantified (n = 3 in each group). Scale bar, 50 μm. ( H ) The above FISCs were IF stained for MyoD expression four hrs after seeding (Left) and the percentage of positively stained cells was quantified (Right) (n = 3 in each group). Scale bar, 50 μm. ( I ) RNAs were isolated from the above SCs cultured for 24 hrs and subject to RNA-seq analysis. Differentially expressed genes were identified with blue and red dots representing down- or up-regulated genes, respectively. ( J - K ) GO analysis of the above up- or down-regulated genes was performed and the top five enriched items are shown in y axis. X axis shows the −log 10 [ P value] of the GO terms. ( L ) Genomic snapshots showing two examples of significantly down-regulated genes in sgMyc vs. ctrl treatment. ( M ) Immunostaining of Pax7 (red) and Laminin (green) waere performed on the uninjured contralateral TA muscle (Left). The number of Pax7 positive cells per 100 fibers was counted (Right) (n = 3 in each group). Scale bar, 50 μm. ( N ) Average fiber size of the above TA muscle was quantified (n = 3 in each group). ( O ) H E staining was performed 5 days post the BaCl 2 injection. Uninjured TA muscle was used as control (Left). The regenerating myofibers with CLN per field was quantified (Right) (n = 3 in each group). Scale bar, 100 μm. ( P ) Immunostaining of eMyHC (red) and Laminin (green) on the above TA muscle sections (Left) and the number of eMyHC+ myofibers was counted (Right) (n = 3 in each group). Scale bar, 50 μm. ( Q ) The cross-sectional area (CSA) of the newly formed fibers with CLN was quantified (n = 3 in each group). All the bar graphs are presented as mean ± s.d. *P

    Techniques Used: CRISPR, Activation Assay, Selection, In Vivo, Mouse Assay, Isolation, Injection, Polymerase Chain Reaction, Cell Culture, Expressing, Quantitative RT-PCR, Western Blot, Labeling, Staining, RNA Sequencing Assay, Immunostaining

    33) Product Images from "Transposase-assisted tagmentation of RNA/DNA hybrid duplexes"

    Article Title: Transposase-assisted tagmentation of RNA/DNA hybrid duplexes

    Journal: eLife

    doi: 10.7554/eLife.54919

    Quality assessment of TRACE-seq. ( a ) Gene expression measured by two technical replicates of TRACE-seq with 20 ng total RNA as input are shown as scatter plots. Pearson's product-moment correlations are displayed in the upper left corner. ( b ) Gene expression measured by two technical replicates of TRACE-seq with 2 ng total RNA as input are shown as scatter plots. Pearson's product-moment correlations are displayed in the upper left corner. ( c ) Scatterplots showing gene expression values for TRACE-seq and NEBNext Ultra II RNA kit with 10 ng mRNA as input (left), and for TRACE-seq with 10 ng mRNA as input and Smart-seq2 with 20 ng total RNA as input (right). All expressed genes (FPKM > 0.5) are included. Pearson's product-moment correlation is displayed in the upper left corner. ( d ) Distribution of the insert size in NEBNext Ultra II RNA library, Smart-seq2 libraries and TRACE-seq libraries with different amount of RNA as input, respectively. ( e ) Median coefficient of variation of gene coverage over the 1000 most highly expressed transcripts in NEBNext Ultra II RNA library, Smart-seq2 libraries and TRACE-seq libraries with different amount of RNA as input, respectively. ( f ) Comparison of read coverage over gene body for NEBNext Ultra II RNA kit, Smart-seq2 and TRACE-seq with different amount of RNA as input. Transcripts were grouped according to annotated lengths and analyzed separately, with the transcript length ranges indicated (top right). The read coverage over gene body is displayed along with gene body percentile from 5’ to 3’ end. ( g ) Library complexity for each library shown by plotting the number of uniquely occurring read-pairs with respect to total number of sampled read-pairs. ( h ) Assessment of RNA Integrity number (RIN). RNA of high RIN score (9.5) was used as input, which allows us to rule out the possibility that the 3’ end bias of gene body coverage is due to RNA degradation. ( i ) Nucleotide versus cycle (NVC) plots showing percentage of observed bases at each position of the first 30 bases of each sequencing read from TRACE-seq library with 10 ng mRNA and 20 ng total RNA as input. ( j ) WebLogo plot of sequence conservations of the first 10 bases of all sequencing reads from TRACE-seq library with 10 ng mRNA as input. The overall height of the stack indicates the sequence conservation at that position (measured in bits), while the height of symbols within the stack indicates the relative frequency of each nucleic acid at that position.
    Figure Legend Snippet: Quality assessment of TRACE-seq. ( a ) Gene expression measured by two technical replicates of TRACE-seq with 20 ng total RNA as input are shown as scatter plots. Pearson's product-moment correlations are displayed in the upper left corner. ( b ) Gene expression measured by two technical replicates of TRACE-seq with 2 ng total RNA as input are shown as scatter plots. Pearson's product-moment correlations are displayed in the upper left corner. ( c ) Scatterplots showing gene expression values for TRACE-seq and NEBNext Ultra II RNA kit with 10 ng mRNA as input (left), and for TRACE-seq with 10 ng mRNA as input and Smart-seq2 with 20 ng total RNA as input (right). All expressed genes (FPKM > 0.5) are included. Pearson's product-moment correlation is displayed in the upper left corner. ( d ) Distribution of the insert size in NEBNext Ultra II RNA library, Smart-seq2 libraries and TRACE-seq libraries with different amount of RNA as input, respectively. ( e ) Median coefficient of variation of gene coverage over the 1000 most highly expressed transcripts in NEBNext Ultra II RNA library, Smart-seq2 libraries and TRACE-seq libraries with different amount of RNA as input, respectively. ( f ) Comparison of read coverage over gene body for NEBNext Ultra II RNA kit, Smart-seq2 and TRACE-seq with different amount of RNA as input. Transcripts were grouped according to annotated lengths and analyzed separately, with the transcript length ranges indicated (top right). The read coverage over gene body is displayed along with gene body percentile from 5’ to 3’ end. ( g ) Library complexity for each library shown by plotting the number of uniquely occurring read-pairs with respect to total number of sampled read-pairs. ( h ) Assessment of RNA Integrity number (RIN). RNA of high RIN score (9.5) was used as input, which allows us to rule out the possibility that the 3’ end bias of gene body coverage is due to RNA degradation. ( i ) Nucleotide versus cycle (NVC) plots showing percentage of observed bases at each position of the first 30 bases of each sequencing read from TRACE-seq library with 10 ng mRNA and 20 ng total RNA as input. ( j ) WebLogo plot of sequence conservations of the first 10 bases of all sequencing reads from TRACE-seq library with 10 ng mRNA as input. The overall height of the stack indicates the sequence conservation at that position (measured in bits), while the height of symbols within the stack indicates the relative frequency of each nucleic acid at that position.

    Techniques Used: Expressing, Sequencing

    Performance of TRACE-seq in differential expression analysis. ( a ) Volcano plot showing differential expressed genes between undifferentiated and differentiated mESCs detected by NEBNext Ultra II RNA kit and TRACE-seq. Significantly up-regulated and down-regulated expressed genes (padj
    Figure Legend Snippet: Performance of TRACE-seq in differential expression analysis. ( a ) Volcano plot showing differential expressed genes between undifferentiated and differentiated mESCs detected by NEBNext Ultra II RNA kit and TRACE-seq. Significantly up-regulated and down-regulated expressed genes (padj

    Techniques Used: Expressing

    34) Product Images from "The RNA Polymerase II core subunit RPB-9 directs transcriptional elongation at piRNA loci in Caenorhabditis elegans"

    Article Title: The RNA Polymerase II core subunit RPB-9 directs transcriptional elongation at piRNA loci in Caenorhabditis elegans

    Journal: bioRxiv

    doi: 10.1101/2020.05.01.070433

    rpb-9 represses two DNA transposon families and a subset of somatic genes. A ) Differential expression analysis of transposable elements in rpb-9 ( mj261 ) mutants versus wild type (polyA-selected RNA-seq libraries). B ) Table showing the numbers of matching piRNAs (with 0, 1, 2 and 3 mismatches respectively) for each of the upregulated transposon families. C ) Genome-wide differential expression analysis of rpb-9 ( mj261 ) mutants versus wild-type (polyA-selected RNA-seq libraries). [p
    Figure Legend Snippet: rpb-9 represses two DNA transposon families and a subset of somatic genes. A ) Differential expression analysis of transposable elements in rpb-9 ( mj261 ) mutants versus wild type (polyA-selected RNA-seq libraries). B ) Table showing the numbers of matching piRNAs (with 0, 1, 2 and 3 mismatches respectively) for each of the upregulated transposon families. C ) Genome-wide differential expression analysis of rpb-9 ( mj261 ) mutants versus wild-type (polyA-selected RNA-seq libraries). [p

    Techniques Used: Expressing, RNA Sequencing Assay, Genome Wide

    35) Product Images from "Enhancers predominantly regulate gene expression in vivo via transcription initiation"

    Article Title: Enhancers predominantly regulate gene expression in vivo via transcription initiation

    Journal: bioRxiv

    doi: 10.1101/844191

    Intronic RNA as a measure of nascent transcription and windowing calculation. Related to Figure 3 . (A) Correlation between intronic RNA coverage over all annotated introns (downloaded from UCSC table browser) comparing Total RNA, polyA+ and polyA-RNA data type and showing all data type correlate extremely highly ( > = 0.94 pearson r 2 ), suggesting they provide similar measures of intronic RNA. All coverage values are a mean of two isogenic duplicates. (B) Meta coverage of introns for each refseq gene. Data from total, polyA+ and polyA-types. Analysis shows that all data types provide comparable coverage over genes. With polyA+ showing the least 5’ coverage and most uniform coverage across genes. All coverage plots are a merge of two isogenic duplicates for each data types. (C) Example gene Eif1 with scaRNA-seq, Total RNA (intronic reads only), polyA+ RNA (intronic reads only) and polyA- (intronic reads only) showing the most uniform coverage is generated from polyA+ RNA. (D) A plot of the distances between observed and annotated TSS, indicating that in primary mouse fetal liver genes observed TSS are systematically found downstream of their annotated positions (as in human (K562 cells). 94% of TSS are found in a window of - 100→+300 relative to the annotated TSS, defining these regions as suitable for counting scaRNA molecules for differential count analysis. (E) Tfdp2 exemplifies increases in intronic RNA and decreases in scaRNA at 0h and 24h across the region of promoter proximal transcription (putative Pol II pausing). Close inspection of the promoter proximal region (grey box marked with red asterisk) reveals a loss of scaRNA signal at the TSS in question (TSS1) but a gain of signal at an alternative upstream TSS (TSS2), arguing against regulation of Pol II pausing.
    Figure Legend Snippet: Intronic RNA as a measure of nascent transcription and windowing calculation. Related to Figure 3 . (A) Correlation between intronic RNA coverage over all annotated introns (downloaded from UCSC table browser) comparing Total RNA, polyA+ and polyA-RNA data type and showing all data type correlate extremely highly ( > = 0.94 pearson r 2 ), suggesting they provide similar measures of intronic RNA. All coverage values are a mean of two isogenic duplicates. (B) Meta coverage of introns for each refseq gene. Data from total, polyA+ and polyA-types. Analysis shows that all data types provide comparable coverage over genes. With polyA+ showing the least 5’ coverage and most uniform coverage across genes. All coverage plots are a merge of two isogenic duplicates for each data types. (C) Example gene Eif1 with scaRNA-seq, Total RNA (intronic reads only), polyA+ RNA (intronic reads only) and polyA- (intronic reads only) showing the most uniform coverage is generated from polyA+ RNA. (D) A plot of the distances between observed and annotated TSS, indicating that in primary mouse fetal liver genes observed TSS are systematically found downstream of their annotated positions (as in human (K562 cells). 94% of TSS are found in a window of - 100→+300 relative to the annotated TSS, defining these regions as suitable for counting scaRNA molecules for differential count analysis. (E) Tfdp2 exemplifies increases in intronic RNA and decreases in scaRNA at 0h and 24h across the region of promoter proximal transcription (putative Pol II pausing). Close inspection of the promoter proximal region (grey box marked with red asterisk) reveals a loss of scaRNA signal at the TSS in question (TSS1) but a gain of signal at an alternative upstream TSS (TSS2), arguing against regulation of Pol II pausing.

    Techniques Used: Generated

    36) Product Images from "Transcriptome-Wide Mapping of RNA 5-Methylcytosine in Arabidopsis mRNAs and Noncoding RNAs"

    Article Title: Transcriptome-Wide Mapping of RNA 5-Methylcytosine in Arabidopsis mRNAs and Noncoding RNAs

    Journal: The Plant Cell

    doi: 10.1105/tpc.16.00751

    Transcriptome-Wide Mapping of m 5 C Using bsRNA-Seq in Arabidopsis. (A) Venn diagram showing the number of m 5 /RBS-seq ( n ≤ 0.3 and ≥1% methylation. (B) Gene schematic and genome browser view of a differentially methylated gene, MAG5 , in three different tissue types. In the MAG5 gene schematic, the methylated cytosine C3349 is indicated. Boxes represent exons; lines represent introns. In the genome browser view, black arrows indicate m 5 C position C3349 (chr5: 19, 262, 126). Top: m 5 reads mapped to the MAG5 locus (chr5: 19, 262, 122-19, 262, 130) from the separate tissue data sets: siliques, shoots, and roots. Each row represents one sequence read and each column a nucleotide. Gray boxes represent nucleotides matching with the MAG5 reference sequence, and red boxes indicate mismatching nucleotides and/or nonmethylated, bisulfite converted cytosines. Sequencing gaps are shown in white. (C) Distribution of m 5 C sites within different RNA types from siliques, shoots, and roots. The numbers in parentheses are percentage of total. (D) Scatterplot showing that increased methylation correlates with lower mRNA abundance in Arabidopsis roots ( r s = −0.32, *P value ≤ 0.0001, Spearman’s correlation). (E) Histogram showing relative enrichment of observed m 5 C sites versus expected number of m 5 across all three tissue types (*P value ≤ 0.0001, binomial test). (F) Metagene profile of m 5 ≤ 0.3 and ≥2% methylation). (G) Methylation of candidate m 5 results. Heat map depicts log arcsine transformed methylation percentages of five m 5 . The numbers in parentheses are genome coordinates, and + or – indicates the strand.
    Figure Legend Snippet: Transcriptome-Wide Mapping of m 5 C Using bsRNA-Seq in Arabidopsis. (A) Venn diagram showing the number of m 5 /RBS-seq ( n ≤ 0.3 and ≥1% methylation. (B) Gene schematic and genome browser view of a differentially methylated gene, MAG5 , in three different tissue types. In the MAG5 gene schematic, the methylated cytosine C3349 is indicated. Boxes represent exons; lines represent introns. In the genome browser view, black arrows indicate m 5 C position C3349 (chr5: 19, 262, 126). Top: m 5 reads mapped to the MAG5 locus (chr5: 19, 262, 122-19, 262, 130) from the separate tissue data sets: siliques, shoots, and roots. Each row represents one sequence read and each column a nucleotide. Gray boxes represent nucleotides matching with the MAG5 reference sequence, and red boxes indicate mismatching nucleotides and/or nonmethylated, bisulfite converted cytosines. Sequencing gaps are shown in white. (C) Distribution of m 5 C sites within different RNA types from siliques, shoots, and roots. The numbers in parentheses are percentage of total. (D) Scatterplot showing that increased methylation correlates with lower mRNA abundance in Arabidopsis roots ( r s = −0.32, *P value ≤ 0.0001, Spearman’s correlation). (E) Histogram showing relative enrichment of observed m 5 C sites versus expected number of m 5 across all three tissue types (*P value ≤ 0.0001, binomial test). (F) Metagene profile of m 5 ≤ 0.3 and ≥2% methylation). (G) Methylation of candidate m 5 results. Heat map depicts log arcsine transformed methylation percentages of five m 5 . The numbers in parentheses are genome coordinates, and + or – indicates the strand.

    Techniques Used: Methylation, Sequencing, Transformation Assay

    37) Product Images from "Probing the sRNA regulatory landscape of P. aeruginosa: post-transcriptional control of determinants of pathogenicity and antibiotic susceptibility"

    Article Title: Probing the sRNA regulatory landscape of P. aeruginosa: post-transcriptional control of determinants of pathogenicity and antibiotic susceptibility

    Journal: Molecular microbiology

    doi: 10.1111/mmi.13857

    Schematic overview of Hi-GRIL-seq. Induction of T4 RNA ligase expression from the P tac promoter with IPTG leads to the expression of the enzyme and the formation of chimeras between base paired endogenous sRNAs and their targets. Following isolation of total RNA and rRNA depletion, a cDNA library for Illumina sequencing is constructed and sequenced. RNA interactions between sRNAs and their targets are identified by a BLAST-based analysis pipeline. Global chimeras are visualized in a two-dimensional dot plot, in which the location of the dot represents the genomic coordinate of the participating RNAs. To examine the targets of a particular RNA, the coverage of its targets can be visualized. To further zoom in on a particular interaction between the two RNAs, the exact location of ligation junctions in the chimeras are mapped and visualized.
    Figure Legend Snippet: Schematic overview of Hi-GRIL-seq. Induction of T4 RNA ligase expression from the P tac promoter with IPTG leads to the expression of the enzyme and the formation of chimeras between base paired endogenous sRNAs and their targets. Following isolation of total RNA and rRNA depletion, a cDNA library for Illumina sequencing is constructed and sequenced. RNA interactions between sRNAs and their targets are identified by a BLAST-based analysis pipeline. Global chimeras are visualized in a two-dimensional dot plot, in which the location of the dot represents the genomic coordinate of the participating RNAs. To examine the targets of a particular RNA, the coverage of its targets can be visualized. To further zoom in on a particular interaction between the two RNAs, the exact location of ligation junctions in the chimeras are mapped and visualized.

    Techniques Used: Expressing, Isolation, cDNA Library Assay, Sequencing, Construct, Ligation

    38) Product Images from "Small RNA-mediated DNA (cytosine-5) methyltransferase 1 inhibition leads to aberrant DNA methylation"

    Article Title: Small RNA-mediated DNA (cytosine-5) methyltransferase 1 inhibition leads to aberrant DNA methylation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv518

    Transcriptome analysis of miR-155-5p and random 23-mers control RNA. ( A ) MA plot analysis of miR-155-5p versus random 23-mers control displaying up- and downregulated genes. ( B ) Normalized read counts of the top five genes with the lowest adjusted P values.
    Figure Legend Snippet: Transcriptome analysis of miR-155-5p and random 23-mers control RNA. ( A ) MA plot analysis of miR-155-5p versus random 23-mers control displaying up- and downregulated genes. ( B ) Normalized read counts of the top five genes with the lowest adjusted P values.

    Techniques Used:

    Correlation of differential transcription and differential genomic methylation. ( A ) Heat map of rlog transformed read counts from top 100 genes with lowest adjusted P values showing the differential transcription of the random 23-mers control RNA and miR-155-5p transfected cells. Correlation of differential methylation of genome and differential transcription by overlapping the gene list obtained from RRBS and RNA-seq dataset. ( B ) All genes covered by RRBS versus whole transcriptome. ( C ) Upregulated genes versus hypomethylated genes. ( D ) Downregulated genes versus hypermethylated genes.
    Figure Legend Snippet: Correlation of differential transcription and differential genomic methylation. ( A ) Heat map of rlog transformed read counts from top 100 genes with lowest adjusted P values showing the differential transcription of the random 23-mers control RNA and miR-155-5p transfected cells. Correlation of differential methylation of genome and differential transcription by overlapping the gene list obtained from RRBS and RNA-seq dataset. ( B ) All genes covered by RRBS versus whole transcriptome. ( C ) Upregulated genes versus hypomethylated genes. ( D ) Downregulated genes versus hypermethylated genes.

    Techniques Used: Methylation, Transformation Assay, Transfection, RNA Sequencing Assay

    39) Product Images from "CRISPR/Cas9 Screens Reveal Multiple Layers of B cell CD40 Regulation"

    Article Title: CRISPR/Cas9 Screens Reveal Multiple Layers of B cell CD40 Regulation

    Journal: Cell reports

    doi: 10.1016/j.celrep.2019.06.079

    The m6A Writer METTL3-METTL14-WTAP and ESCRT Negatively Regulates CD40 Abundance (A) Log2-normlized CRISPR screen mean + SD abundances are shown for sgRNAs targeting the gene encoding METTL14. Mean + SD of two input libraries and four screen replicates are shown. (B) FACS analysis of PM Fas abundances in Cas9 + Daudi B cells expressing control or METTL14-targeting sgRNAs and stimulated by 50 ng/mL Mega-CD40L for 48 h, as indicated. (C) Immunoblots analysis of CD40, METTL14, or control tubulin abundances in WCE from Cas9 + Daudi B cells expressing the indicated sgRNAs. (D) Immunoblots analysis of CD40, IκBα, or GAPDH abundances in Cas9 + Daudi B cells expressing the indicated sgRNAs pre-treated for 1 h with 5 μM Calbiochem IKK inhibitor VIII (IKK inh) and then treated with IKK inh together with 50 ng/mL Mega-CD40L for an additional 12 h, as indicated. (E) RT-PCR analysis of 18S-rRNA normalized CD40 mRNA levels in Cas9 + Daudi B cells expressing the indicated sgRNA. (F) RT-qPCR analysis of Daudi B cell CD40 mRNA immunopurified by control IgG versus IgG against WTAP component VIRMA, expressed as a percentage of WCE input CD40 RNA abundance. (G) RT-qPCR analysis of CD40 abundances in control IgG versus anti-m6A mRNA immunoprecipitations from Cas9 + Daudi B cells that expressed control or METTL14 sgRNAs. Immunopurified CD40 mRNA abundances are expressed as a percentage of input CD40 mRNA levels. (H) Schematic highlighting ESCRT component CD40 negative regulator screen hits. ESCRT-0, II, and III subunits hits included genes encoding HRS, VPS25,VPS36, CHMP5, and CHMP6. (I) Log2-normalized CRISPR screen CHMP5 and VPS25 sgRNA abundances. Mean + SD of two input libraries and four screen replicates are shown. (J) FACS analysis of PM Fas abundance in Cas9 + Daudi B cells with the indicated control, CHMP5 , or VPS25 sgRNAs and stimulated with 50 ng/mL Mega-CD40L for 48 h, as indicated. (K) FACS analysis of PM CD40 abundances in Cas9 + Daudi B cells with the indicated control, CHMP5 , or VPS25 sgRNAs and stimulated with 50 ng/mL Mega-CD40L for 24 h, as indicated. (L) PM MFI Fas abundances as in (K) from n = 3 experiments. (M) Immunoblot analysis of WCE from Cas9 + Daudi B cells expressing the indicated control or either of two independent CHMP5 or VPS25 targeting sgRNAs and stimulated for 24 h with 50 ng/mL Mega-CD40L, as indicated. Mean + SD from n = 3 are shown in (E)–(G), (J), and (L). *p
    Figure Legend Snippet: The m6A Writer METTL3-METTL14-WTAP and ESCRT Negatively Regulates CD40 Abundance (A) Log2-normlized CRISPR screen mean + SD abundances are shown for sgRNAs targeting the gene encoding METTL14. Mean + SD of two input libraries and four screen replicates are shown. (B) FACS analysis of PM Fas abundances in Cas9 + Daudi B cells expressing control or METTL14-targeting sgRNAs and stimulated by 50 ng/mL Mega-CD40L for 48 h, as indicated. (C) Immunoblots analysis of CD40, METTL14, or control tubulin abundances in WCE from Cas9 + Daudi B cells expressing the indicated sgRNAs. (D) Immunoblots analysis of CD40, IκBα, or GAPDH abundances in Cas9 + Daudi B cells expressing the indicated sgRNAs pre-treated for 1 h with 5 μM Calbiochem IKK inhibitor VIII (IKK inh) and then treated with IKK inh together with 50 ng/mL Mega-CD40L for an additional 12 h, as indicated. (E) RT-PCR analysis of 18S-rRNA normalized CD40 mRNA levels in Cas9 + Daudi B cells expressing the indicated sgRNA. (F) RT-qPCR analysis of Daudi B cell CD40 mRNA immunopurified by control IgG versus IgG against WTAP component VIRMA, expressed as a percentage of WCE input CD40 RNA abundance. (G) RT-qPCR analysis of CD40 abundances in control IgG versus anti-m6A mRNA immunoprecipitations from Cas9 + Daudi B cells that expressed control or METTL14 sgRNAs. Immunopurified CD40 mRNA abundances are expressed as a percentage of input CD40 mRNA levels. (H) Schematic highlighting ESCRT component CD40 negative regulator screen hits. ESCRT-0, II, and III subunits hits included genes encoding HRS, VPS25,VPS36, CHMP5, and CHMP6. (I) Log2-normalized CRISPR screen CHMP5 and VPS25 sgRNA abundances. Mean + SD of two input libraries and four screen replicates are shown. (J) FACS analysis of PM Fas abundance in Cas9 + Daudi B cells with the indicated control, CHMP5 , or VPS25 sgRNAs and stimulated with 50 ng/mL Mega-CD40L for 48 h, as indicated. (K) FACS analysis of PM CD40 abundances in Cas9 + Daudi B cells with the indicated control, CHMP5 , or VPS25 sgRNAs and stimulated with 50 ng/mL Mega-CD40L for 24 h, as indicated. (L) PM MFI Fas abundances as in (K) from n = 3 experiments. (M) Immunoblot analysis of WCE from Cas9 + Daudi B cells expressing the indicated control or either of two independent CHMP5 or VPS25 targeting sgRNAs and stimulated for 24 h with 50 ng/mL Mega-CD40L, as indicated. Mean + SD from n = 3 are shown in (E)–(G), (J), and (L). *p

    Techniques Used: CRISPR, FACS, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

    40) Product Images from "Network Walking charts transcriptional dynamics of nitrogen signaling by integrating validated and predicted genome-wide interactions"

    Article Title: Network Walking charts transcriptional dynamics of nitrogen signaling by integrating validated and predicted genome-wide interactions

    Journal: Nature Communications

    doi: 10.1038/s41467-019-09522-1

    Network Walking connects validated direct transcription factor (TF) targets to in planta responses. a Schematic overview: the Network Walking approach charts a network path from direct targets of a TF identified in cells to its indirect targets, which only respond in planta. This is achieved using data for 33 TF perturbations in root cells using TARGET (Transient Assay Reporting Genome-wide Effects of Transcription factors) 18 scaled-up in this study, and a time-series transcriptome of nitrogen (N) response in whole roots 29 . TF–target edges for 145 TFs were inferred using this time-series data in a machine-learning method called dynamic factor graphs (DFG) 48 (blue arrow). The inferred edges were pruned for high-confidence edges (purple arrow) using 71,836 validated edges (red arrow) for 33 TFs in a precision/recall analysis (area under precision recall (AUPR)). The validated edges and high-confidence inferred edges are used to link a TF to its indirect targets in planta via the Network Walk. b The 33 TFs were selected based on their response to N in shoots and roots (black TFs) or roots only (orange TFs) from the N-treatment time-series data of Varala et al. 29 . TFs were placed into groups based on their Just-in-Time classification, in which genes were binned based on the first time-point N treatment caused a fold change of 1.5 relative to the control 29 . TFs in bold have been previously described in the nitrogen response (CRF4 and CDF1 29 , NAC4 30 , TGA1 and TGA4 26 , 27 , LBD37 and LBD38 31 , HHO2 and HHO3 32 ). Asterisk indicates TFs not included in the DFG network as they did not meet the false discovery rate (FDR) threshold
    Figure Legend Snippet: Network Walking connects validated direct transcription factor (TF) targets to in planta responses. a Schematic overview: the Network Walking approach charts a network path from direct targets of a TF identified in cells to its indirect targets, which only respond in planta. This is achieved using data for 33 TF perturbations in root cells using TARGET (Transient Assay Reporting Genome-wide Effects of Transcription factors) 18 scaled-up in this study, and a time-series transcriptome of nitrogen (N) response in whole roots 29 . TF–target edges for 145 TFs were inferred using this time-series data in a machine-learning method called dynamic factor graphs (DFG) 48 (blue arrow). The inferred edges were pruned for high-confidence edges (purple arrow) using 71,836 validated edges (red arrow) for 33 TFs in a precision/recall analysis (area under precision recall (AUPR)). The validated edges and high-confidence inferred edges are used to link a TF to its indirect targets in planta via the Network Walk. b The 33 TFs were selected based on their response to N in shoots and roots (black TFs) or roots only (orange TFs) from the N-treatment time-series data of Varala et al. 29 . TFs were placed into groups based on their Just-in-Time classification, in which genes were binned based on the first time-point N treatment caused a fold change of 1.5 relative to the control 29 . TFs in bold have been previously described in the nitrogen response (CRF4 and CDF1 29 , NAC4 30 , TGA1 and TGA4 26 , 27 , LBD37 and LBD38 31 , HHO2 and HHO3 32 ). Asterisk indicates TFs not included in the DFG network as they did not meet the false discovery rate (FDR) threshold

    Techniques Used: Genome Wide

    Related Articles

    Amplification:

    Article Title: KDM5 inhibition offers a novel therapeutic strategy for the treatment of KMT2D mutant lymphomas
    Article Snippet: .. Briefly, ChIP and input DNA were end-repaired, adaptors ligated and size-selected using SPRIselect beads for 300-400bp DNA fragments and amplified by PCR. ..

    Article Title: Symmetric neural progenitor divisions require chromatin-mediated homologous recombination DNA repair by Ino80
    Article Snippet: .. After PCR amplification, libraries were purified with the 1.2 × AMPure Beads. .. Purified ATACseq libraries were analyzed for quality and nucleosome periodicity using a BioAnalyzer High Sensitivity DNA chip (Agilent Technologies) and quantified using the NEBNext Library Quant Kit for Illumina.

    Article Title: RNA Sequencing by Direct Tagmentation of RNA/DNA Hybrids
    Article Snippet: .. When performing high-throughput experiment, each sample could be amplified by 15 cycles, then merged for beads purification and library quality check. .. As for purified 10ng or 200ng total RNA input, the tagmentation product was firstly gap-filled with 100 units of Superscript II and 1 x Q5 High-Fidelity Master Mix at 42°C for 15min, then Superscript II was inactivated at 70°C for 15min.

    Isolation:

    Article Title: Anopheles mosquitoes revealed new principles of 3D genome organization in insects
    Article Snippet: .. Samples were prepared for Illumina sequencing with NEBNext® Ultra™ II RNA Library Prep Kit for Illumina (NEB #E7775) accompanied by NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) with RNA insert size of 200 bp. .. Ovary preservation and polytene chromosome preparation To prepare high-quality polytene chromosome slides we followed the protocol described in Sharakhova et al. with minor exceptions.

    Spectrophotometry:

    Article Title: Advanced Safety and Genetic Stability in Mice of a Novel DNA-Launched Venezuelan Equine Encephalitis Virus Vaccine with Rearranged Structural Genes
    Article Snippet: .. Purified RNA was quantified on a NanoDrop 2000 spectrophotometer (ThermoFisher, Wilmington, DE, USA) Samples were prepared for sequencing by rRNA depletion using the NEBNext rRNA Depletion Kit (#E6310) followed by library prep using NEBNEXT Ultra II RNA Library Prep with Sample Purification Beads (#E7775, NEB, Ipswich, MA, USA). .. Libraries were quality checked on an Agilent BioAnalyzer before sequencing on an Illumina NextSeq 500 with a NextSeq 500/550 High Output Kit v2.5 (75 Cycles) kit (#20024906, Illumina, San Diego, CA, USA).

    Purification:

    Article Title: Advanced Safety and Genetic Stability in Mice of a Novel DNA-Launched Venezuelan Equine Encephalitis Virus Vaccine with Rearranged Structural Genes
    Article Snippet: .. Purified RNA was quantified on a NanoDrop 2000 spectrophotometer (ThermoFisher, Wilmington, DE, USA) Samples were prepared for sequencing by rRNA depletion using the NEBNext rRNA Depletion Kit (#E6310) followed by library prep using NEBNEXT Ultra II RNA Library Prep with Sample Purification Beads (#E7775, NEB, Ipswich, MA, USA). .. Libraries were quality checked on an Agilent BioAnalyzer before sequencing on an Illumina NextSeq 500 with a NextSeq 500/550 High Output Kit v2.5 (75 Cycles) kit (#20024906, Illumina, San Diego, CA, USA).

    Article Title: Symmetric neural progenitor divisions require chromatin-mediated homologous recombination DNA repair by Ino80
    Article Snippet: .. After PCR amplification, libraries were purified with the 1.2 × AMPure Beads. .. Purified ATACseq libraries were analyzed for quality and nucleosome periodicity using a BioAnalyzer High Sensitivity DNA chip (Agilent Technologies) and quantified using the NEBNext Library Quant Kit for Illumina.

    Article Title: RNA Sequencing by Direct Tagmentation of RNA/DNA Hybrids
    Article Snippet: .. When performing high-throughput experiment, each sample could be amplified by 15 cycles, then merged for beads purification and library quality check. .. As for purified 10ng or 200ng total RNA input, the tagmentation product was firstly gap-filled with 100 units of Superscript II and 1 x Q5 High-Fidelity Master Mix at 42°C for 15min, then Superscript II was inactivated at 70°C for 15min.

    Article Title: Replication timing maintains the global epigenetic state in human cells
    Article Snippet: .. Excess adaptors were removed using an additional 0.9x AMPure bead purification. .. KAPA qPCR (KAPA Biosystems) was used to determine molar quantities of each library and individual libraries were diluted and pooled at equimolar concentrations.

    Sequencing:

    Article Title: Anopheles mosquitoes revealed new principles of 3D genome organization in insects
    Article Snippet: .. Samples were prepared for Illumina sequencing with NEBNext® Ultra™ II RNA Library Prep Kit for Illumina (NEB #E7775) accompanied by NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) with RNA insert size of 200 bp. .. Ovary preservation and polytene chromosome preparation To prepare high-quality polytene chromosome slides we followed the protocol described in Sharakhova et al. with minor exceptions.

    Article Title: Advanced Safety and Genetic Stability in Mice of a Novel DNA-Launched Venezuelan Equine Encephalitis Virus Vaccine with Rearranged Structural Genes
    Article Snippet: .. Purified RNA was quantified on a NanoDrop 2000 spectrophotometer (ThermoFisher, Wilmington, DE, USA) Samples were prepared for sequencing by rRNA depletion using the NEBNext rRNA Depletion Kit (#E6310) followed by library prep using NEBNEXT Ultra II RNA Library Prep with Sample Purification Beads (#E7775, NEB, Ipswich, MA, USA). .. Libraries were quality checked on an Agilent BioAnalyzer before sequencing on an Illumina NextSeq 500 with a NextSeq 500/550 High Output Kit v2.5 (75 Cycles) kit (#20024906, Illumina, San Diego, CA, USA).

    Polymerase Chain Reaction:

    Article Title: KDM5 inhibition offers a novel therapeutic strategy for the treatment of KMT2D mutant lymphomas
    Article Snippet: .. Briefly, ChIP and input DNA were end-repaired, adaptors ligated and size-selected using SPRIselect beads for 300-400bp DNA fragments and amplified by PCR. ..

    Article Title: Symmetric neural progenitor divisions require chromatin-mediated homologous recombination DNA repair by Ino80
    Article Snippet: .. After PCR amplification, libraries were purified with the 1.2 × AMPure Beads. .. Purified ATACseq libraries were analyzed for quality and nucleosome periodicity using a BioAnalyzer High Sensitivity DNA chip (Agilent Technologies) and quantified using the NEBNext Library Quant Kit for Illumina.

    other:

    Article Title: Screening and identification of miRNAs related to sexual differentiation of strobili in Ginkgo biloba by integration analysis of small RNA, RNA, and degradome sequencing
    Article Snippet: Degradome library construction Total RNA from the female and male strobili of G. biloba on March 13 and April 5 was mixed for the construction of the degradome library according to the kit instruction of NEBNext Ultra II RNA Library Prep Kit (NEB, E7775, USA).

    High Throughput Screening Assay:

    Article Title: RNA Sequencing by Direct Tagmentation of RNA/DNA Hybrids
    Article Snippet: .. When performing high-throughput experiment, each sample could be amplified by 15 cycles, then merged for beads purification and library quality check. .. As for purified 10ng or 200ng total RNA input, the tagmentation product was firstly gap-filled with 100 units of Superscript II and 1 x Q5 High-Fidelity Master Mix at 42°C for 15min, then Superscript II was inactivated at 70°C for 15min.

    Chromatin Immunoprecipitation:

    Article Title: KDM5 inhibition offers a novel therapeutic strategy for the treatment of KMT2D mutant lymphomas
    Article Snippet: .. Briefly, ChIP and input DNA were end-repaired, adaptors ligated and size-selected using SPRIselect beads for 300-400bp DNA fragments and amplified by PCR. ..

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    New England Biolabs nebnext ultra rna library prep kit for illumina
    Nebnext Ultra Rna Library Prep Kit For Illumina, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nebnext ultra rna library prep kit for illumina/product/New England Biolabs
    Average 99 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    nebnext ultra rna library prep kit for illumina - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs nebnext ultra directional rna library prep kit for illumina
    Nebnext Ultra Directional Rna Library Prep Kit For Illumina, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 474 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nebnext ultra directional rna library prep kit for illumina/product/New England Biolabs
    Average 99 stars, based on 474 article reviews
    Price from $9.99 to $1999.99
    nebnext ultra directional rna library prep kit for illumina - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs ultra directional rna seq kit
    Ultra Directional Rna Seq Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ultra directional rna seq kit/product/New England Biolabs
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    ultra directional rna seq kit - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    Image Search Results