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  • 99
    New England Biolabs ultra directional rna library kit
    Internode zones have unique <t>RNAseq</t> profiles and contain stem- and zone-specific genes. a Principal component analysis of FPKM values obtained from <t>RNA</t> sequencing of the MsZ, CEZ, TZ, and MZ of internode 5. b Venn diagram of genes up-regulated greater than a log 2 fold change of one against the background levels in the whole plant (WP) transcriptome. c Table showing the number of genes expressed > 1 FPKM, number of genes up-regulated against the WP transcriptome with > 1 log 2 fold, number of identified ‘stem-specific’ genes with > 2.5 log 2 fold change against the WP background and > 80 FPKM in any internode zone, and the number of ‘zone-specific’ genes with, in addition to being ‘stem-specific’, > 2 log 2 fold change against all other internodal zones (with some exceptions, see Additional file 2 )
    Ultra Directional Rna Library Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1018 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc ultra directional rna library prep kit
    Internode zones have unique <t>RNAseq</t> profiles and contain stem- and zone-specific genes. a Principal component analysis of FPKM values obtained from <t>RNA</t> sequencing of the MsZ, CEZ, TZ, and MZ of internode 5. b Venn diagram of genes up-regulated greater than a log 2 fold change of one against the background levels in the whole plant (WP) transcriptome. c Table showing the number of genes expressed > 1 FPKM, number of genes up-regulated against the WP transcriptome with > 1 log 2 fold, number of identified ‘stem-specific’ genes with > 2.5 log 2 fold change against the WP background and > 80 FPKM in any internode zone, and the number of ‘zone-specific’ genes with, in addition to being ‘stem-specific’, > 2 log 2 fold change against all other internodal zones (with some exceptions, see Additional file 2 )
    Ultra Directional Rna Library Prep Kit, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 98/100, based on 176 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs ultra rna library kit
    Internode zones have unique <t>RNAseq</t> profiles and contain stem- and zone-specific genes. a Principal component analysis of FPKM values obtained from <t>RNA</t> sequencing of the MsZ, CEZ, TZ, and MZ of internode 5. b Venn diagram of genes up-regulated greater than a log 2 fold change of one against the background levels in the whole plant (WP) transcriptome. c Table showing the number of genes expressed > 1 FPKM, number of genes up-regulated against the WP transcriptome with > 1 log 2 fold, number of identified ‘stem-specific’ genes with > 2.5 log 2 fold change against the WP background and > 80 FPKM in any internode zone, and the number of ‘zone-specific’ genes with, in addition to being ‘stem-specific’, > 2 log 2 fold change against all other internodal zones (with some exceptions, see Additional file 2 )
    Ultra Rna Library Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 184 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs ultratm ii directional rna library prep kit
    Internode zones have unique <t>RNAseq</t> profiles and contain stem- and zone-specific genes. a Principal component analysis of FPKM values obtained from <t>RNA</t> sequencing of the MsZ, CEZ, TZ, and MZ of internode 5. b Venn diagram of genes up-regulated greater than a log 2 fold change of one against the background levels in the whole plant (WP) transcriptome. c Table showing the number of genes expressed > 1 FPKM, number of genes up-regulated against the WP transcriptome with > 1 log 2 fold, number of identified ‘stem-specific’ genes with > 2.5 log 2 fold change against the WP background and > 80 FPKM in any internode zone, and the number of ‘zone-specific’ genes with, in addition to being ‘stem-specific’, > 2 log 2 fold change against all other internodal zones (with some exceptions, see Additional file 2 )
    Ultratm Ii Directional Rna Library Prep Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs rna library prep kit
    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 <t>RNA</t> 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 <t>mRNA</t> 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
    Rna Library Prep Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 330 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs nebnext ultra ii directional rna library prep with sample purification beads
    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 <t>RNA</t> 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 <t>mRNA</t> 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
    Nebnext Ultra Ii Directional Rna Library Prep With Sample Purification Beads, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc illumina next generation sequencing
    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 <t>RNA</t> 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 <t>mRNA</t> 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
    Illumina Next Generation Sequencing, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 1175 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs rrna depletion kit
    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 <t>RNA</t> 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 <t>mRNA</t> 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
    Rrna Depletion Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Internode zones have unique RNAseq profiles and contain stem- and zone-specific genes. a Principal component analysis of FPKM values obtained from RNA sequencing of the MsZ, CEZ, TZ, and MZ of internode 5. b Venn diagram of genes up-regulated greater than a log 2 fold change of one against the background levels in the whole plant (WP) transcriptome. c Table showing the number of genes expressed > 1 FPKM, number of genes up-regulated against the WP transcriptome with > 1 log 2 fold, number of identified ‘stem-specific’ genes with > 2.5 log 2 fold change against the WP background and > 80 FPKM in any internode zone, and the number of ‘zone-specific’ genes with, in addition to being ‘stem-specific’, > 2 log 2 fold change against all other internodal zones (with some exceptions, see Additional file 2 )

    Journal: Biotechnology for Biofuels

    Article Title: A developing Setaria viridis internode: an experimental system for the study of biomass generation in a C4 model species

    doi: 10.1186/s13068-016-0457-6

    Figure Lengend Snippet: Internode zones have unique RNAseq profiles and contain stem- and zone-specific genes. a Principal component analysis of FPKM values obtained from RNA sequencing of the MsZ, CEZ, TZ, and MZ of internode 5. b Venn diagram of genes up-regulated greater than a log 2 fold change of one against the background levels in the whole plant (WP) transcriptome. c Table showing the number of genes expressed > 1 FPKM, number of genes up-regulated against the WP transcriptome with > 1 log 2 fold, number of identified ‘stem-specific’ genes with > 2.5 log 2 fold change against the WP background and > 80 FPKM in any internode zone, and the number of ‘zone-specific’ genes with, in addition to being ‘stem-specific’, > 2 log 2 fold change against all other internodal zones (with some exceptions, see Additional file 2 )

    Article Snippet: RNAseq libraries were generated with polyA enrichment and rRNA depletion using NEBNext® Ultra™ Directional RNA Library Prep Kit (NEB, Ipswich, USA, http://www.neb.com ) for Illumina® and were sequenced on an Illumina® HiSeq 2500 at the Max Planck Genome Centre, Cologne, Germany.

    Techniques: RNA Sequencing Assay

    KMT2D loss leads to reduced keratinocyte proliferation and a broad loss of epithelial development and adhesion genes. ( A ) KMT2D mRNA expression is significantly reduced in both NHEKs ( P = 0.0036) and HaCaTs ( P = 0.0312) treated with shKMT2D as determined by RNA-seq. ( B ) shKMT2D keratinocytes display reduced levels of KMT2D but normal-appearing nuclear morphology. ( C ) shKMT2D keratinocytes display reduced proliferation in comparison with shSC keratinocytes. ( D ) Representative genes from shKMTD NHEKs that are significantly reduced in expression along with corresponding adjusted P -values. ( E ) Gene ontology (GO) analysis of the 134 common genes lost with shKMT2D treatment between both shKMT2D NHEKs and HaCaTs demonstrates that genes involved in key epithelial signaling pathways ( RARG and VDR ), epithelial cell growth and morphogenesis, polarity, and adhesion are enriched among those genes with reduced expression. ( F ) shKMT2D keratinocytes display reduced expression of RARγ and VDR by IF (40×).

    Journal: Genes & Development

    Article Title: KMT2D regulates p63 target enhancers to coordinate epithelial homeostasis

    doi: 10.1101/gad.306241.117

    Figure Lengend Snippet: KMT2D loss leads to reduced keratinocyte proliferation and a broad loss of epithelial development and adhesion genes. ( A ) KMT2D mRNA expression is significantly reduced in both NHEKs ( P = 0.0036) and HaCaTs ( P = 0.0312) treated with shKMT2D as determined by RNA-seq. ( B ) shKMT2D keratinocytes display reduced levels of KMT2D but normal-appearing nuclear morphology. ( C ) shKMT2D keratinocytes display reduced proliferation in comparison with shSC keratinocytes. ( D ) Representative genes from shKMTD NHEKs that are significantly reduced in expression along with corresponding adjusted P -values. ( E ) Gene ontology (GO) analysis of the 134 common genes lost with shKMT2D treatment between both shKMT2D NHEKs and HaCaTs demonstrates that genes involved in key epithelial signaling pathways ( RARG and VDR ), epithelial cell growth and morphogenesis, polarity, and adhesion are enriched among those genes with reduced expression. ( F ) shKMT2D keratinocytes display reduced expression of RARγ and VDR by IF (40×).

    Article Snippet: All RNA-seq libraries were prepared using the NEBNext poly(A) mRNA magnetic isolation module followed by NEBNext Ultra Directional RNA library preparation kit for Illumina (both from New England Biolabs).

    Techniques: Expressing, RNA Sequencing Assay

    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

    Journal: Genes & Development

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

    doi: 10.1101/gad.241950.114

    Figure Lengend 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

    Article Snippet: An NEBNext ultradirectional RNA library kit (New England Biolabs, catalog no. E7420S) was used to make cDNA and RNA-seq libraries.

    Techniques: RNA Sequencing Assay, Derivative Assay

    Dynamics of H3K4me3 and expression in subsets of genes. ( A ) Genomic regions subject to age-linked CNV detected by comparison between input samples; these were excluded from ChIP analysis. ( B ) Scatter plot of log 2 -transformed mRNAseq read counts from protein coding genes defined in Figure 4A normalised for ORF length comparing log phase cells to 48-hr-aged cells. Data averaged across five replicates per condition. ( C ) Average profile of H3K4me3 ChIP signal for 1 kb either side of the TSS performed as in Figure 4C , for 300 genes least induced with age. ( D ) As ( C ) for 300 most induced genes. ( E ) Hierarchical clustering analysis of log 2 -transformed mRNA levels across age for the 4806 unambiguous genes defined in Figure 4A .

    Journal: eLife

    Article Title: Tri-methylation of histone H3 lysine 4 facilitates gene expression in ageing cells

    doi: 10.7554/eLife.34081

    Figure Lengend Snippet: Dynamics of H3K4me3 and expression in subsets of genes. ( A ) Genomic regions subject to age-linked CNV detected by comparison between input samples; these were excluded from ChIP analysis. ( B ) Scatter plot of log 2 -transformed mRNAseq read counts from protein coding genes defined in Figure 4A normalised for ORF length comparing log phase cells to 48-hr-aged cells. Data averaged across five replicates per condition. ( C ) Average profile of H3K4me3 ChIP signal for 1 kb either side of the TSS performed as in Figure 4C , for 300 genes least induced with age. ( D ) As ( C ) for 300 most induced genes. ( E ) Hierarchical clustering analysis of log 2 -transformed mRNA levels across age for the 4806 unambiguous genes defined in Figure 4A .

    Article Snippet: 500 ng RNA was used to prepare libraries using the NEBNext Ultra Directional mRNAseq kit (NEB E7420, E7490, E7335, E7500, E7710) as described with modifications: Reagent volumes for RT, second strand synthesis, tailing and ligation were reduced by 50%, while libraries were amplified for 12 cycles using 2 µl each primer per reaction before two rounds of AMPure bead purification as described in the kit at 0.9x ratio prior to quality control using a Bioanalyzer and quantification usinng KAPA Biosystems kit KK4824.

    Techniques: Expressing, Chromatin Immunoprecipitation, Transformation Assay

    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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-019-08345-4

    Figure Lengend 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

    Article Snippet: RNA-seq libraries were prepared from the mRNA using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs).

    Techniques: 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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-019-08345-4

    Figure Lengend 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

    Article Snippet: RNA-seq libraries were prepared from the mRNA using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs).

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

    RNA knockdown activity screen of engineered Cas13d orthologs in human cells (A) Schematic for mammalian expression constructs encoding for engineered Cas13d effectors and guides. NLS, nuclear localization signal. pre-gRNA, artificial unprocessed guide RNA containing a single 30 nt spacer sequence flanked by 2 full length 36 nt DRs. gRNA, predicted mature guide RNA with a single 30 nt processed DR and 22 nt spacer sequence (B) Heatmap of mCherry protein knockdown in a Cas13d ortholog activity screen in HEK 293FT cells using pools of 4 pre-gRNAs or gRNAs. Normalized MFI, median fluorescent intensity relative to non-targeting condition. Positions in gray were not tested, with n = 3. (C) Immunocytochemistry of Cas13d showing localization and expression of engineered constructs. Scale bar, 10 μm. Blue pseudocolor, DAPI staining of nuclei. (D) Comparison of Adm and Rfx Cas13d ortholog constructs for knockdown of endogenous B4GALNT1 mRNA reveals RfxCas13d-NLS (CasRx) to be most effective for both guide RNA architectures. Pools of 4 guides were used for targeting. NT, non-targeting. Values are mean ± SEM with n = 3.

    Journal: Cell

    Article Title: Transcriptome engineering with RNA-targeting Type VI-D CRISPR effectors

    doi: 10.1016/j.cell.2018.02.033

    Figure Lengend Snippet: RNA knockdown activity screen of engineered Cas13d orthologs in human cells (A) Schematic for mammalian expression constructs encoding for engineered Cas13d effectors and guides. NLS, nuclear localization signal. pre-gRNA, artificial unprocessed guide RNA containing a single 30 nt spacer sequence flanked by 2 full length 36 nt DRs. gRNA, predicted mature guide RNA with a single 30 nt processed DR and 22 nt spacer sequence (B) Heatmap of mCherry protein knockdown in a Cas13d ortholog activity screen in HEK 293FT cells using pools of 4 pre-gRNAs or gRNAs. Normalized MFI, median fluorescent intensity relative to non-targeting condition. Positions in gray were not tested, with n = 3. (C) Immunocytochemistry of Cas13d showing localization and expression of engineered constructs. Scale bar, 10 μm. Blue pseudocolor, DAPI staining of nuclei. (D) Comparison of Adm and Rfx Cas13d ortholog constructs for knockdown of endogenous B4GALNT1 mRNA reveals RfxCas13d-NLS (CasRx) to be most effective for both guide RNA architectures. Pools of 4 guides were used for targeting. NT, non-targeting. Values are mean ± SEM with n = 3.

    Article Snippet: Stranded mRNA libraries were prepared using the NEBNext II Ultra Directional RNA Library Prep Kit from New England Biolabs (Cat# E7760S) and sequenced on an Illumina NextSeq500 with 42 nt paired end reads.

    Techniques: Activity Assay, Expressing, Construct, Sequencing, Immunocytochemistry, Staining