globin sequencing Search Results


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  • 92
    Millipore rabbit globin mrna
    Analysis of MosIR expression and phenotype. ( A ) Schematic composition of the MosIR transgene. ( B ) A schematic structure of the MosIR transcript folded into a long dsRNA hairpin. The length of the stem is 520 bp. ( C ) MosIR transgene produces EGFP in somatic tissues. From left to right: brain, kidney, liver, spleen, tail. All images were taken with the same settings. No autofluorescence was visible under these conditions in internal organs of wild-type mice. ( D ) MosIR transgene (+) shows normal segregation into male and female progeny in crosses of MosIR males and wild-type female mice. ( E ) MosIR positive females phenocopy the Mos null phenotype: parthenogenetic activation of ovulated eggs. The left panel shows normal metaphase II-arrested eggs. The right panel shows transgenic parthenogenotes with two extruded polar bodies (black arrowheads) and cleaving parthenogenotes (white arrowheads). ( F ) qPCR of Mos <t>mRNA</t> in fully-grown oocytes obtained from three transgenic animals reveals strong reduction of Mos mRNA level compared to wild-type oocytes. Error bars = SEM. ( G ) <t>RNase</t> T1-resistant MosIR RNA can be detected in MEFs isolated from MosIR embryos and in 293 cells transfected with the MosIR plasmid. Hypotonically lysed cells were treated with RNase T1 for 30 min at 37°C before RNA extraction (T1). The efficiency of RNase T1 digestion was tested by disrupting secondary RNA structures by heat before RNase T1 digestion (heat+T1). RT indicates reverse-transcribed RNA (+) and controls without reverse transcriptase (−). cDNA was amplified with MosIR-specific primers for 31 cycles (MEF) and 30 cycles (293) of PCR. No amplification was observed with primers located in the single-stranded EGFP coding sequence (data not shown).
    Rabbit Globin Mrna, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Illumina Inc globin zerotm
    Analysis of MosIR expression and phenotype. ( A ) Schematic composition of the MosIR transgene. ( B ) A schematic structure of the MosIR transcript folded into a long dsRNA hairpin. The length of the stem is 520 bp. ( C ) MosIR transgene produces EGFP in somatic tissues. From left to right: brain, kidney, liver, spleen, tail. All images were taken with the same settings. No autofluorescence was visible under these conditions in internal organs of wild-type mice. ( D ) MosIR transgene (+) shows normal segregation into male and female progeny in crosses of MosIR males and wild-type female mice. ( E ) MosIR positive females phenocopy the Mos null phenotype: parthenogenetic activation of ovulated eggs. The left panel shows normal metaphase II-arrested eggs. The right panel shows transgenic parthenogenotes with two extruded polar bodies (black arrowheads) and cleaving parthenogenotes (white arrowheads). ( F ) qPCR of Mos <t>mRNA</t> in fully-grown oocytes obtained from three transgenic animals reveals strong reduction of Mos mRNA level compared to wild-type oocytes. Error bars = SEM. ( G ) <t>RNase</t> T1-resistant MosIR RNA can be detected in MEFs isolated from MosIR embryos and in 293 cells transfected with the MosIR plasmid. Hypotonically lysed cells were treated with RNase T1 for 30 min at 37°C before RNA extraction (T1). The efficiency of RNase T1 digestion was tested by disrupting secondary RNA structures by heat before RNase T1 digestion (heat+T1). RT indicates reverse-transcribed RNA (+) and controls without reverse transcriptase (−). cDNA was amplified with MosIR-specific primers for 31 cycles (MEF) and 30 cycles (293) of PCR. No amplification was observed with primers located in the single-stranded EGFP coding sequence (data not shown).
    Globin Zerotm, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/globin zerotm/product/Illumina Inc
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    85
    TaKaRa rabbit globin poly a sequence
    Analysis of MosIR expression and phenotype. ( A ) Schematic composition of the MosIR transgene. ( B ) A schematic structure of the MosIR transcript folded into a long dsRNA hairpin. The length of the stem is 520 bp. ( C ) MosIR transgene produces EGFP in somatic tissues. From left to right: brain, kidney, liver, spleen, tail. All images were taken with the same settings. No autofluorescence was visible under these conditions in internal organs of wild-type mice. ( D ) MosIR transgene (+) shows normal segregation into male and female progeny in crosses of MosIR males and wild-type female mice. ( E ) MosIR positive females phenocopy the Mos null phenotype: parthenogenetic activation of ovulated eggs. The left panel shows normal metaphase II-arrested eggs. The right panel shows transgenic parthenogenotes with two extruded polar bodies (black arrowheads) and cleaving parthenogenotes (white arrowheads). ( F ) qPCR of Mos <t>mRNA</t> in fully-grown oocytes obtained from three transgenic animals reveals strong reduction of Mos mRNA level compared to wild-type oocytes. Error bars = SEM. ( G ) <t>RNase</t> T1-resistant MosIR RNA can be detected in MEFs isolated from MosIR embryos and in 293 cells transfected with the MosIR plasmid. Hypotonically lysed cells were treated with RNase T1 for 30 min at 37°C before RNA extraction (T1). The efficiency of RNase T1 digestion was tested by disrupting secondary RNA structures by heat before RNase T1 digestion (heat+T1). RT indicates reverse-transcribed RNA (+) and controls without reverse transcriptase (−). cDNA was amplified with MosIR-specific primers for 31 cycles (MEF) and 30 cycles (293) of PCR. No amplification was observed with primers located in the single-stranded EGFP coding sequence (data not shown).
    Rabbit Globin Poly A Sequence, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher direct α globin gene sequencing
    Analysis of MosIR expression and phenotype. ( A ) Schematic composition of the MosIR transgene. ( B ) A schematic structure of the MosIR transcript folded into a long dsRNA hairpin. The length of the stem is 520 bp. ( C ) MosIR transgene produces EGFP in somatic tissues. From left to right: brain, kidney, liver, spleen, tail. All images were taken with the same settings. No autofluorescence was visible under these conditions in internal organs of wild-type mice. ( D ) MosIR transgene (+) shows normal segregation into male and female progeny in crosses of MosIR males and wild-type female mice. ( E ) MosIR positive females phenocopy the Mos null phenotype: parthenogenetic activation of ovulated eggs. The left panel shows normal metaphase II-arrested eggs. The right panel shows transgenic parthenogenotes with two extruded polar bodies (black arrowheads) and cleaving parthenogenotes (white arrowheads). ( F ) qPCR of Mos <t>mRNA</t> in fully-grown oocytes obtained from three transgenic animals reveals strong reduction of Mos mRNA level compared to wild-type oocytes. Error bars = SEM. ( G ) <t>RNase</t> T1-resistant MosIR RNA can be detected in MEFs isolated from MosIR embryos and in 293 cells transfected with the MosIR plasmid. Hypotonically lysed cells were treated with RNase T1 for 30 min at 37°C before RNA extraction (T1). The efficiency of RNase T1 digestion was tested by disrupting secondary RNA structures by heat before RNase T1 digestion (heat+T1). RT indicates reverse-transcribed RNA (+) and controls without reverse transcriptase (−). cDNA was amplified with MosIR-specific primers for 31 cycles (MEF) and 30 cycles (293) of PCR. No amplification was observed with primers located in the single-stranded EGFP coding sequence (data not shown).
    Direct α Globin Gene Sequencing, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    OriGene β globin cdna
    Targeting therapeutic <t>β-globin</t> <t>cDNA</t> to the endogenous β-globin locus. ( A ) β-in-frame-cDNA targeting vector. When targeted, the cDNA is expressed from the endogenous ATG start site. pA, BGH polyadenylation signal sequence; Ubc, Ubiquitin C promoter; MGMT, Methylguanine methyltransferase. ( B ) Schematic of the wt (top) and targeted (bottom) β-globin locus. The absence of intron 1 in the cDNA-targeted locus results in a shorter PCR product. ( C ) Results from SMRT sequencing of targeted β-globin alleles without selection (top row) and with up to three rounds of drug selection.
    β Globin Cdna, supplied by OriGene, used in various techniques. Bioz Stars score: 85/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Abcam γ globin antibody
    miR-138-5p targets BCL11A and regulates <t>γ-globin</t> expression. ( a ) Top10 down-regulated miRNA candidates in the plasma exosomes of β-TM as determined by NGS analysis. ( b ) Predicted miR-138-5p binding site in the 3′UTR of BCL11A with sequence complementarity. ( c ) Schema of reporter vector (pmirGLO) carrying either WT or mutant 3′UTR of BCL11A. Luciferase assays using a reporter vector (pmirGLO) carrying either WT or mutant 3′UTR of BCL11A in K562 cells transfected with scramble control microRNA mimics (controls, 100 nM), miR-138-5p mimics (100 nM) for 24 hrs. Experiments were performed in triplicates. The values represented are mean ± SD, one-way ANOVA, ****P
    γ Globin Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    ViennaLab Diagnostics β globin stripassay sea
    miR-138-5p targets BCL11A and regulates <t>γ-globin</t> expression. ( a ) Top10 down-regulated miRNA candidates in the plasma exosomes of β-TM as determined by NGS analysis. ( b ) Predicted miR-138-5p binding site in the 3′UTR of BCL11A with sequence complementarity. ( c ) Schema of reporter vector (pmirGLO) carrying either WT or mutant 3′UTR of BCL11A. Luciferase assays using a reporter vector (pmirGLO) carrying either WT or mutant 3′UTR of BCL11A in K562 cells transfected with scramble control microRNA mimics (controls, 100 nM), miR-138-5p mimics (100 nM) for 24 hrs. Experiments were performed in triplicates. The values represented are mean ± SD, one-way ANOVA, ****P
    β Globin Stripassay Sea, supplied by ViennaLab Diagnostics, used in various techniques. Bioz Stars score: 85/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher native globin mrna
    Activities of eIF1 mutants in 48S complex formation. Toeprinting analysis of 48S complexes assembled on β-globin <t>mRNA</t> in the presence of 40S subunits, eIF2, eIF3, eIF4A, eIF4B, eIF4F, <t>eIF1A,</t> aminoacylated initiator tRNA, and the N-terminally truncated eIF1 Δ29 mutant ( A ) or Fe(II)-BABE-derivatized cysteineless and single-cysteine eIF1 mutants ( B ) as indicated. Reaction mixtures containing only β-globin mRNA are shown in lane 1 of each panel. Reaction mixtures corresponding to lane 2 of each panel contained all translation components except eIF1. Reference lanes A and G depict 18S rRNA sequence generated using the same primer. Complex I is arrested 21–24 nt from the 5′-end of β-globin mRNA. The 48S complex assembled on the AUG triplet is designated as “complex II (+15–17 nt from AUG).” The 48S complex assembled on the GUG triplet located 23 nt from the 5′-end of β-globin mRNA is designated as “+16 nt from GUG.”
    Native Globin Mrna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher β globin mrna
    Toeprint analysis of 48S ribosomal complex formation on <t>β-globin</t> <t>mRNA</t> with recombinant eIF4GI. The components of each reaction mixture are indicated above the lanes. Formation of complex II was quantified as described in Materials and Methods, and the value for eIF4F (lane 2) was set at 100.
    β Globin Mrna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Marker Gene Technologies wild type β globin gene sequence
    Elevation of γ-Globin Expression by Genome Editing Nucleases targeted to genomic sites create double-strand breaks (DSBs) that can be repaired by non-homologous end joining (NHEJ) or by homology-directed repair (HDR). Shown on the left are three examples for deletions causing increases in fetal hemoglobin production. The first shows the creation of a 13-kb deletion encompassing the δ- and <t>β-globin</t> gene. This deletion removes negative regulatory elements and positions the β-globin 3′ enhancer in close proximity to the γ-globin genes. The second example shows deletion of a repressor binding site in the γ-globin gene promoters. The third example shows creation of a frameshift in the coding region or a deletion of a positive DNA-regulatory element in one of the BLC11A erythroid-specific enhancers. Shown on the right are two examples for HDR-mediated correction of the sickle cell mutation. The first example shows an adeno-associated vector (AAV) that delivers the homologous arms (HAs), the therapeutic donor DNA, and a selectable marker (ITR: inverted terminal repeats). The second example shows a single stranded oligodeoxynucleotide containing 5′ and 3′ homologous arms (HAs) and the wild-type β-globin sequence. The diagram on the bottom right outlines the major steps involved in genome editing using autologous cell transplantation.
    Wild Type β Globin Gene Sequence, supplied by Marker Gene Technologies, used in various techniques. Bioz Stars score: 92/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Illumina Inc globin transcripts
    Elevation of γ-Globin Expression by Genome Editing Nucleases targeted to genomic sites create double-strand breaks (DSBs) that can be repaired by non-homologous end joining (NHEJ) or by homology-directed repair (HDR). Shown on the left are three examples for deletions causing increases in fetal hemoglobin production. The first shows the creation of a 13-kb deletion encompassing the δ- and <t>β-globin</t> gene. This deletion removes negative regulatory elements and positions the β-globin 3′ enhancer in close proximity to the γ-globin genes. The second example shows deletion of a repressor binding site in the γ-globin gene promoters. The third example shows creation of a frameshift in the coding region or a deletion of a positive DNA-regulatory element in one of the BLC11A erythroid-specific enhancers. Shown on the right are two examples for HDR-mediated correction of the sickle cell mutation. The first example shows an adeno-associated vector (AAV) that delivers the homologous arms (HAs), the therapeutic donor DNA, and a selectable marker (ITR: inverted terminal repeats). The second example shows a single stranded oligodeoxynucleotide containing 5′ and 3′ homologous arms (HAs) and the wild-type β-globin sequence. The diagram on the bottom right outlines the major steps involved in genome editing using autologous cell transplantation.
    Globin Transcripts, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 89/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Sigma-Genosys β globin
    Cleavage site preferences for falcipain-2 and falcipain-3. Preferences for amino acids (single letter code) at P 1 -P 4  and P 1 ′-P 4 ′ are shown schematically. At each position, the height of each amino acid corresponds to its frequency at cleavage sites based on analysis of α and β globin. WebLogo ( weblogo.berkeley.edu ), a free software for plotting consensus sequences, was used to plot P 4-  P 4 ′ preferences.
    β Globin, supplied by Sigma-Genosys, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    New England Biolabs nebnext globin
    Cleavage site preferences for falcipain-2 and falcipain-3. Preferences for amino acids (single letter code) at P 1 -P 4  and P 1 ′-P 4 ′ are shown schematically. At each position, the height of each amino acid corresponds to its frequency at cleavage sites based on analysis of α and β globin. WebLogo ( weblogo.berkeley.edu ), a free software for plotting consensus sequences, was used to plot P 4-  P 4 ′ preferences.
    Nebnext Globin, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Stratagene α globin
    Yield of various truncated α-Hbs after purification. The different soluble fractions obtained after disruption of induced cells was applied on Glutathione Sepharose 4B in PBS (150 mM NaCl, 10 mM Na 2 HPO 4 , pH 7.4). After washing, various truncated GST-AHSP WT /GST-α-Hb complexes were eluted (elution buffer: 50 mM Tris-HCl, 20 mM reduced glutathione pH 8.0) and analyzed by (A) SDS-PAGE (12%) and (B) Western Blotting using anti-α globin antibodies.
    α Globin, supplied by Stratagene, used in various techniques. Bioz Stars score: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Illumina Inc ribo zero globin
    Experimental Design and Analysis. ( a ) Blood <t>RNA</t> preparation steps from 6 human subjects: 3 controls ( c ) and 3 patients (P). RNA was used for preparing libraries with <t>Ribo-Zero</t> Gold (RZG) or Globin-Zero (GZ) kits as shown. In Experiment 1, a total of 33 libraries were prepared with either 250 ng total RNA for low (L) input or 900 ng for high (H) input. In Experiment 2 technical replicate libraries were prepared using 250 ng RNA from C1 and C2 with either RZG or Globin-Zero for a total of 12 libraries. Individual RNA-seq data files generated from the libraries shown in B and C were named according to the following convention: sample ID_RNA input_library method_replicate number, e.g., C1_L_GZ_1. ( b ) Fastq files were assessed for quality metrics, aligned and analyzed as shown. Correspondence between data analysis steps and results shown in Figs. 2 – 4 is indicated.
    Ribo Zero Globin, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 62 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Illumina Inc globin zero gold kit
    Experimental Design and Analysis. ( a ) Blood <t>RNA</t> preparation steps from 6 human subjects: 3 controls ( c ) and 3 patients (P). RNA was used for preparing libraries with <t>Ribo-Zero</t> Gold (RZG) or Globin-Zero (GZ) kits as shown. In Experiment 1, a total of 33 libraries were prepared with either 250 ng total RNA for low (L) input or 900 ng for high (H) input. In Experiment 2 technical replicate libraries were prepared using 250 ng RNA from C1 and C2 with either RZG or Globin-Zero for a total of 12 libraries. Individual RNA-seq data files generated from the libraries shown in B and C were named according to the following convention: sample ID_RNA input_library method_replicate number, e.g., C1_L_GZ_1. ( b ) Fastq files were assessed for quality metrics, aligned and analyzed as shown. Correspondence between data analysis steps and results shown in Figs. 2 – 4 is indicated.
    Globin Zero Gold Kit, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 91/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Illumina Inc ribo zero globin kit
    Experimental Design and Analysis. ( a ) Blood <t>RNA</t> preparation steps from 6 human subjects: 3 controls ( c ) and 3 patients (P). RNA was used for preparing libraries with <t>Ribo-Zero</t> Gold (RZG) or Globin-Zero (GZ) kits as shown. In Experiment 1, a total of 33 libraries were prepared with either 250 ng total RNA for low (L) input or 900 ng for high (H) input. In Experiment 2 technical replicate libraries were prepared using 250 ng RNA from C1 and C2 with either RZG or Globin-Zero for a total of 12 libraries. Individual RNA-seq data files generated from the libraries shown in B and C were named according to the following convention: sample ID_RNA input_library method_replicate number, e.g., C1_L_GZ_1. ( b ) Fastq files were assessed for quality metrics, aligned and analyzed as shown. Correspondence between data analysis steps and results shown in Figs. 2 – 4 is indicated.
    Ribo Zero Globin Kit, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 91/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    FRITSCH GmbH human globin related sequence psi beta 2
    Experimental Design and Analysis. ( a ) Blood <t>RNA</t> preparation steps from 6 human subjects: 3 controls ( c ) and 3 patients (P). RNA was used for preparing libraries with <t>Ribo-Zero</t> Gold (RZG) or Globin-Zero (GZ) kits as shown. In Experiment 1, a total of 33 libraries were prepared with either 250 ng total RNA for low (L) input or 900 ng for high (H) input. In Experiment 2 technical replicate libraries were prepared using 250 ng RNA from C1 and C2 with either RZG or Globin-Zero for a total of 12 libraries. Individual RNA-seq data files generated from the libraries shown in B and C were named according to the following convention: sample ID_RNA input_library method_replicate number, e.g., C1_L_GZ_1. ( b ) Fastq files were assessed for quality metrics, aligned and analyzed as shown. Correspondence between data analysis steps and results shown in Figs. 2 – 4 is indicated.
    Human Globin Related Sequence Psi Beta 2, supplied by FRITSCH GmbH, used in various techniques. Bioz Stars score: 80/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Illumina Inc globin mrna
    Experimental Design and Analysis. ( a ) Blood <t>RNA</t> preparation steps from 6 human subjects: 3 controls ( c ) and 3 patients (P). RNA was used for preparing libraries with <t>Ribo-Zero</t> Gold (RZG) or Globin-Zero (GZ) kits as shown. In Experiment 1, a total of 33 libraries were prepared with either 250 ng total RNA for low (L) input or 900 ng for high (H) input. In Experiment 2 technical replicate libraries were prepared using 250 ng RNA from C1 and C2 with either RZG or Globin-Zero for a total of 12 libraries. Individual RNA-seq data files generated from the libraries shown in B and C were named according to the following convention: sample ID_RNA input_library method_replicate number, e.g., C1_L_GZ_1. ( b ) Fastq files were assessed for quality metrics, aligned and analyzed as shown. Correspondence between data analysis steps and results shown in Figs. 2 – 4 is indicated.
    Globin Mrna, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 90/100, based on 60 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher globin clear kit
    Experimental Design and Analysis. ( a ) Blood <t>RNA</t> preparation steps from 6 human subjects: 3 controls ( c ) and 3 patients (P). RNA was used for preparing libraries with <t>Ribo-Zero</t> Gold (RZG) or Globin-Zero (GZ) kits as shown. In Experiment 1, a total of 33 libraries were prepared with either 250 ng total RNA for low (L) input or 900 ng for high (H) input. In Experiment 2 technical replicate libraries were prepared using 250 ng RNA from C1 and C2 with either RZG or Globin-Zero for a total of 12 libraries. Individual RNA-seq data files generated from the libraries shown in B and C were named according to the following convention: sample ID_RNA input_library method_replicate number, e.g., C1_L_GZ_1. ( b ) Fastq files were assessed for quality metrics, aligned and analyzed as shown. Correspondence between data analysis steps and results shown in Figs. 2 – 4 is indicated.
    Globin Clear Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher globin depletion protocol
    Experimental Design and Analysis. ( a ) Blood <t>RNA</t> preparation steps from 6 human subjects: 3 controls ( c ) and 3 patients (P). RNA was used for preparing libraries with <t>Ribo-Zero</t> Gold (RZG) or Globin-Zero (GZ) kits as shown. In Experiment 1, a total of 33 libraries were prepared with either 250 ng total RNA for low (L) input or 900 ng for high (H) input. In Experiment 2 technical replicate libraries were prepared using 250 ng RNA from C1 and C2 with either RZG or Globin-Zero for a total of 12 libraries. Individual RNA-seq data files generated from the libraries shown in B and C were named according to the following convention: sample ID_RNA input_library method_replicate number, e.g., C1_L_GZ_1. ( b ) Fastq files were assessed for quality metrics, aligned and analyzed as shown. Correspondence between data analysis steps and results shown in Figs. 2 – 4 is indicated.
    Globin Depletion Protocol, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Roche riboerase hmr globin
    Experimental Design and Analysis. ( a ) Blood <t>RNA</t> preparation steps from 6 human subjects: 3 controls ( c ) and 3 patients (P). RNA was used for preparing libraries with <t>Ribo-Zero</t> Gold (RZG) or Globin-Zero (GZ) kits as shown. In Experiment 1, a total of 33 libraries were prepared with either 250 ng total RNA for low (L) input or 900 ng for high (H) input. In Experiment 2 technical replicate libraries were prepared using 250 ng RNA from C1 and C2 with either RZG or Globin-Zero for a total of 12 libraries. Individual RNA-seq data files generated from the libraries shown in B and C were named according to the following convention: sample ID_RNA input_library method_replicate number, e.g., C1_L_GZ_1. ( b ) Fastq files were assessed for quality metrics, aligned and analyzed as shown. Correspondence between data analysis steps and results shown in Figs. 2 – 4 is indicated.
    Riboerase Hmr Globin, supplied by Roche, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc β globin reporter gene plasmid
    Human <t>β-globin</t> reporter gene. ( A ) Detail of the reporter construct (See ‘Materials and methods’). Top schematic drawn to scale. ( B ) Imaging of gene expression from nascent transcripts (arrow) to protein product (blue channel shows CFP-SKL protein product of the reporter gene accumulating in peroxisomes). Images are maximum intensity projections of z-stacks (Δz = 0.25 μm, exposure = 1 s). ( C ) Splicing efficiency and ( D ) poly(A) tail site/length of the β-globin reporter, measured in three conditions: mock-transfected cells and cells transfected with either wild type (WT) or mutant (S34F) splicing factor U2AF1 (see Materials and methods). Error bars are SEM calculated over four measurements. ( E ) Expected fluorescence time profiles for a single transcript. When the PP7 cassette is transcribed, the red fluorescence signal increases progressively (as RNA stem loops are formed) and plateaus once the polymerase exits the cassette. The same applies to the green fluorescence signal when the PP7 cassette is transcribed. If splicing is post-release, red and green signals drop simultaneously when the unspliced RNA is released and diffuse away. If splicing is pre-release, the red fluorescence drops before the green fluorescence reflecting that intron removal occurred before the release of a spliced transcript. DOI: http://dx.doi.org/10.7554/eLife.03939.004
    β Globin Reporter Gene Plasmid, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 86/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Incyte 584 nt mouse β 1 globin mrna sequence
    Human <t>β-globin</t> reporter gene. ( A ) Detail of the reporter construct (See ‘Materials and methods’). Top schematic drawn to scale. ( B ) Imaging of gene expression from nascent transcripts (arrow) to protein product (blue channel shows CFP-SKL protein product of the reporter gene accumulating in peroxisomes). Images are maximum intensity projections of z-stacks (Δz = 0.25 μm, exposure = 1 s). ( C ) Splicing efficiency and ( D ) poly(A) tail site/length of the β-globin reporter, measured in three conditions: mock-transfected cells and cells transfected with either wild type (WT) or mutant (S34F) splicing factor U2AF1 (see Materials and methods). Error bars are SEM calculated over four measurements. ( E ) Expected fluorescence time profiles for a single transcript. When the PP7 cassette is transcribed, the red fluorescence signal increases progressively (as RNA stem loops are formed) and plateaus once the polymerase exits the cassette. The same applies to the green fluorescence signal when the PP7 cassette is transcribed. If splicing is post-release, red and green signals drop simultaneously when the unspliced RNA is released and diffuse away. If splicing is pre-release, the red fluorescence drops before the green fluorescence reflecting that intron removal occurred before the release of a spliced transcript. DOI: http://dx.doi.org/10.7554/eLife.03939.004
    584 Nt Mouse β 1 Globin Mrna Sequence, supplied by Incyte, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Analysis of MosIR expression and phenotype. ( A ) Schematic composition of the MosIR transgene. ( B ) A schematic structure of the MosIR transcript folded into a long dsRNA hairpin. The length of the stem is 520 bp. ( C ) MosIR transgene produces EGFP in somatic tissues. From left to right: brain, kidney, liver, spleen, tail. All images were taken with the same settings. No autofluorescence was visible under these conditions in internal organs of wild-type mice. ( D ) MosIR transgene (+) shows normal segregation into male and female progeny in crosses of MosIR males and wild-type female mice. ( E ) MosIR positive females phenocopy the Mos null phenotype: parthenogenetic activation of ovulated eggs. The left panel shows normal metaphase II-arrested eggs. The right panel shows transgenic parthenogenotes with two extruded polar bodies (black arrowheads) and cleaving parthenogenotes (white arrowheads). ( F ) qPCR of Mos mRNA in fully-grown oocytes obtained from three transgenic animals reveals strong reduction of Mos mRNA level compared to wild-type oocytes. Error bars = SEM. ( G ) RNase T1-resistant MosIR RNA can be detected in MEFs isolated from MosIR embryos and in 293 cells transfected with the MosIR plasmid. Hypotonically lysed cells were treated with RNase T1 for 30 min at 37°C before RNA extraction (T1). The efficiency of RNase T1 digestion was tested by disrupting secondary RNA structures by heat before RNase T1 digestion (heat+T1). RT indicates reverse-transcribed RNA (+) and controls without reverse transcriptase (−). cDNA was amplified with MosIR-specific primers for 31 cycles (MEF) and 30 cycles (293) of PCR. No amplification was observed with primers located in the single-stranded EGFP coding sequence (data not shown).

    Journal: Nucleic Acids Research

    Article Title: dsRNA expression in the mouse elicits RNAi in oocytes and low adenosine deamination in somatic cells

    doi: 10.1093/nar/gkr702

    Figure Lengend Snippet: Analysis of MosIR expression and phenotype. ( A ) Schematic composition of the MosIR transgene. ( B ) A schematic structure of the MosIR transcript folded into a long dsRNA hairpin. The length of the stem is 520 bp. ( C ) MosIR transgene produces EGFP in somatic tissues. From left to right: brain, kidney, liver, spleen, tail. All images were taken with the same settings. No autofluorescence was visible under these conditions in internal organs of wild-type mice. ( D ) MosIR transgene (+) shows normal segregation into male and female progeny in crosses of MosIR males and wild-type female mice. ( E ) MosIR positive females phenocopy the Mos null phenotype: parthenogenetic activation of ovulated eggs. The left panel shows normal metaphase II-arrested eggs. The right panel shows transgenic parthenogenotes with two extruded polar bodies (black arrowheads) and cleaving parthenogenotes (white arrowheads). ( F ) qPCR of Mos mRNA in fully-grown oocytes obtained from three transgenic animals reveals strong reduction of Mos mRNA level compared to wild-type oocytes. Error bars = SEM. ( G ) RNase T1-resistant MosIR RNA can be detected in MEFs isolated from MosIR embryos and in 293 cells transfected with the MosIR plasmid. Hypotonically lysed cells were treated with RNase T1 for 30 min at 37°C before RNA extraction (T1). The efficiency of RNase T1 digestion was tested by disrupting secondary RNA structures by heat before RNase T1 digestion (heat+T1). RT indicates reverse-transcribed RNA (+) and controls without reverse transcriptase (−). cDNA was amplified with MosIR-specific primers for 31 cycles (MEF) and 30 cycles (293) of PCR. No amplification was observed with primers located in the single-stranded EGFP coding sequence (data not shown).

    Article Snippet: RNA isolation and quantitative real-time RT–PCR Oocytes for real-time PCR analysis were washed 3-times in PBS, placed individually in pure water with 10 U of RiboLock RNase inhibitor (Fermentas) while 0.14 pg/µl of rabbit globin mRNA (Sigma Aldrich) and 0.2 µg/µl of E. coli rRNA (Roche) was added to act as an external standard and to inhibit sample RNA degradation by RNases.

    Techniques: Expressing, Mouse Assay, Activation Assay, Transgenic Assay, Real-time Polymerase Chain Reaction, Isolation, Transfection, Plasmid Preparation, RNA Extraction, Amplification, Polymerase Chain Reaction, Sequencing

    Targeting therapeutic β-globin cDNA to the endogenous β-globin locus. ( A ) β-in-frame-cDNA targeting vector. When targeted, the cDNA is expressed from the endogenous ATG start site. pA, BGH polyadenylation signal sequence; Ubc, Ubiquitin C promoter; MGMT, Methylguanine methyltransferase. ( B ) Schematic of the wt (top) and targeted (bottom) β-globin locus. The absence of intron 1 in the cDNA-targeted locus results in a shorter PCR product. ( C ) Results from SMRT sequencing of targeted β-globin alleles without selection (top row) and with up to three rounds of drug selection.

    Journal: Nucleic Acids Research

    Article Title: Nuclease-mediated gene editing by homologous recombination of the human globin locus

    doi: 10.1093/nar/gkt947

    Figure Lengend Snippet: Targeting therapeutic β-globin cDNA to the endogenous β-globin locus. ( A ) β-in-frame-cDNA targeting vector. When targeted, the cDNA is expressed from the endogenous ATG start site. pA, BGH polyadenylation signal sequence; Ubc, Ubiquitin C promoter; MGMT, Methylguanine methyltransferase. ( B ) Schematic of the wt (top) and targeted (bottom) β-globin locus. The absence of intron 1 in the cDNA-targeted locus results in a shorter PCR product. ( C ) Results from SMRT sequencing of targeted β-globin alleles without selection (top row) and with up to three rounds of drug selection.

    Article Snippet: The β-in-frame-cDNA and β-in-frame-GFP targeting vectors were synthesized by overlap PCR to insert β-globin cDNA (OriGene) or GFP directly in-frame to the β-globin ATG start codon using the primers in Supplementary Figure S10 .

    Techniques: Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Selection

    miR-138-5p targets BCL11A and regulates γ-globin expression. ( a ) Top10 down-regulated miRNA candidates in the plasma exosomes of β-TM as determined by NGS analysis. ( b ) Predicted miR-138-5p binding site in the 3′UTR of BCL11A with sequence complementarity. ( c ) Schema of reporter vector (pmirGLO) carrying either WT or mutant 3′UTR of BCL11A. Luciferase assays using a reporter vector (pmirGLO) carrying either WT or mutant 3′UTR of BCL11A in K562 cells transfected with scramble control microRNA mimics (controls, 100 nM), miR-138-5p mimics (100 nM) for 24 hrs. Experiments were performed in triplicates. The values represented are mean ± SD, one-way ANOVA, ****P

    Journal: Scientific Reports

    Article Title: Reciprocal regulation of γ-globin expression by exo-miRNAs: Relevance to γ-globin silencing in β-thalassemia major

    doi: 10.1038/s41598-017-00150-7

    Figure Lengend Snippet: miR-138-5p targets BCL11A and regulates γ-globin expression. ( a ) Top10 down-regulated miRNA candidates in the plasma exosomes of β-TM as determined by NGS analysis. ( b ) Predicted miR-138-5p binding site in the 3′UTR of BCL11A with sequence complementarity. ( c ) Schema of reporter vector (pmirGLO) carrying either WT or mutant 3′UTR of BCL11A. Luciferase assays using a reporter vector (pmirGLO) carrying either WT or mutant 3′UTR of BCL11A in K562 cells transfected with scramble control microRNA mimics (controls, 100 nM), miR-138-5p mimics (100 nM) for 24 hrs. Experiments were performed in triplicates. The values represented are mean ± SD, one-way ANOVA, ****P

    Article Snippet: CD63 antibody (1:500) (NKI/C-3, catalogue no # MA5-11501) was purchased from ThermoScientific. γ-globin antibody was purchased from Abcam (1:1000) (EPR9708, catalogue no ab137096). β-actin antibody was purchased from Sigma-Aldrich (1:4000) (AC-15, catalogue no A5441).

    Techniques: Expressing, Next-Generation Sequencing, Binding Assay, Sequencing, Plasmid Preparation, Mutagenesis, Luciferase, Transfection

    Exo-miRNA mediated γ-globin regulation. Reciprocally expressed exo-miRNAs (miR-223 and miR-138) in the healthy donor and β-TM are involved in the γ-globin regulation.

    Journal: Scientific Reports

    Article Title: Reciprocal regulation of γ-globin expression by exo-miRNAs: Relevance to γ-globin silencing in β-thalassemia major

    doi: 10.1038/s41598-017-00150-7

    Figure Lengend Snippet: Exo-miRNA mediated γ-globin regulation. Reciprocally expressed exo-miRNAs (miR-223 and miR-138) in the healthy donor and β-TM are involved in the γ-globin regulation.

    Article Snippet: CD63 antibody (1:500) (NKI/C-3, catalogue no # MA5-11501) was purchased from ThermoScientific. γ-globin antibody was purchased from Abcam (1:1000) (EPR9708, catalogue no ab137096). β-actin antibody was purchased from Sigma-Aldrich (1:4000) (AC-15, catalogue no A5441).

    Techniques:

    miR-223-3p down regulates LMO2 and γ-globin expression. ( a ) Top10 up-regulated miRNA candidates in the plasma exosomes of β-TM as determined by NGS analysis. ( b ) Predicted miR-223-3p binding site in the 3′UTR of LMO2 with sequence complementarity. ( c ) Schema of a reporter vector (pmirGLO) carrying either WT or mutant 3′UTR of LMO2. Luciferase assays using reporter vector (pmirGLO) carrying either WT or mutant 3′UTR of LMO2 in K562 cells transfected with scramble control microRNA mimics (controls, 100 nM), miR-223-3p mimics (100 nM) or anti-miR-223-3p (antagomir, 100 nM) for 24 hr. Experiments were performed in triplicates. The values represented are mean ± SD, one-way ANOVA, ****P

    Journal: Scientific Reports

    Article Title: Reciprocal regulation of γ-globin expression by exo-miRNAs: Relevance to γ-globin silencing in β-thalassemia major

    doi: 10.1038/s41598-017-00150-7

    Figure Lengend Snippet: miR-223-3p down regulates LMO2 and γ-globin expression. ( a ) Top10 up-regulated miRNA candidates in the plasma exosomes of β-TM as determined by NGS analysis. ( b ) Predicted miR-223-3p binding site in the 3′UTR of LMO2 with sequence complementarity. ( c ) Schema of a reporter vector (pmirGLO) carrying either WT or mutant 3′UTR of LMO2. Luciferase assays using reporter vector (pmirGLO) carrying either WT or mutant 3′UTR of LMO2 in K562 cells transfected with scramble control microRNA mimics (controls, 100 nM), miR-223-3p mimics (100 nM) or anti-miR-223-3p (antagomir, 100 nM) for 24 hr. Experiments were performed in triplicates. The values represented are mean ± SD, one-way ANOVA, ****P

    Article Snippet: CD63 antibody (1:500) (NKI/C-3, catalogue no # MA5-11501) was purchased from ThermoScientific. γ-globin antibody was purchased from Abcam (1:1000) (EPR9708, catalogue no ab137096). β-actin antibody was purchased from Sigma-Aldrich (1:4000) (AC-15, catalogue no A5441).

    Techniques: Expressing, Next-Generation Sequencing, Binding Assay, Sequencing, Plasmid Preparation, Mutagenesis, Luciferase, Transfection

    Exosomes reciprocally regulate γ-globin expressions. ( a ) Schematic representation of exosome isolation, exo-miRNA validation and γ-globin regulation. ( b ) Immunoblot analysis of p-AKT, p-p38 levels in K562 cells treated with β-TM or HD plasma exosomes. Experiments were performed in triplicates. The values represented are mean ± SD, one-way ANOVA, ***P

    Journal: Scientific Reports

    Article Title: Reciprocal regulation of γ-globin expression by exo-miRNAs: Relevance to γ-globin silencing in β-thalassemia major

    doi: 10.1038/s41598-017-00150-7

    Figure Lengend Snippet: Exosomes reciprocally regulate γ-globin expressions. ( a ) Schematic representation of exosome isolation, exo-miRNA validation and γ-globin regulation. ( b ) Immunoblot analysis of p-AKT, p-p38 levels in K562 cells treated with β-TM or HD plasma exosomes. Experiments were performed in triplicates. The values represented are mean ± SD, one-way ANOVA, ***P

    Article Snippet: CD63 antibody (1:500) (NKI/C-3, catalogue no # MA5-11501) was purchased from ThermoScientific. γ-globin antibody was purchased from Abcam (1:1000) (EPR9708, catalogue no ab137096). β-actin antibody was purchased from Sigma-Aldrich (1:4000) (AC-15, catalogue no A5441).

    Techniques: Isolation

    miR-223-3p targeting up regulates γ-globin expression. ( a ) Overview of the barcoded lentiviral miRNA over expression and sponge vector. miRNA expression is driven by the viral CMV promoter. This vector contains the miRNA and ~500 bp endogenous flanking sequences in the 3′UTR of the RFP (Red Fluorescent Protein). To allow selection of target cells, we incorporated a dual selection cassette of puromycin gene. ( b ) RT-qPCR validation for miR-223-3p over expression and sponge repression in K562 cells. ( c ) Western blot analysis of γ-globin expression by viral miR-223-3p over expression or sponge repression in K562 cells. Experiments were performed in triplicates. The values represented are mean ± SD, t-test analysis, *P

    Journal: Scientific Reports

    Article Title: Reciprocal regulation of γ-globin expression by exo-miRNAs: Relevance to γ-globin silencing in β-thalassemia major

    doi: 10.1038/s41598-017-00150-7

    Figure Lengend Snippet: miR-223-3p targeting up regulates γ-globin expression. ( a ) Overview of the barcoded lentiviral miRNA over expression and sponge vector. miRNA expression is driven by the viral CMV promoter. This vector contains the miRNA and ~500 bp endogenous flanking sequences in the 3′UTR of the RFP (Red Fluorescent Protein). To allow selection of target cells, we incorporated a dual selection cassette of puromycin gene. ( b ) RT-qPCR validation for miR-223-3p over expression and sponge repression in K562 cells. ( c ) Western blot analysis of γ-globin expression by viral miR-223-3p over expression or sponge repression in K562 cells. Experiments were performed in triplicates. The values represented are mean ± SD, t-test analysis, *P

    Article Snippet: CD63 antibody (1:500) (NKI/C-3, catalogue no # MA5-11501) was purchased from ThermoScientific. γ-globin antibody was purchased from Abcam (1:1000) (EPR9708, catalogue no ab137096). β-actin antibody was purchased from Sigma-Aldrich (1:4000) (AC-15, catalogue no A5441).

    Techniques: Expressing, Over Expression, Plasmid Preparation, Selection, Quantitative RT-PCR, Western Blot

    Activities of eIF1 mutants in 48S complex formation. Toeprinting analysis of 48S complexes assembled on β-globin mRNA in the presence of 40S subunits, eIF2, eIF3, eIF4A, eIF4B, eIF4F, eIF1A, aminoacylated initiator tRNA, and the N-terminally truncated eIF1 Δ29 mutant ( A ) or Fe(II)-BABE-derivatized cysteineless and single-cysteine eIF1 mutants ( B ) as indicated. Reaction mixtures containing only β-globin mRNA are shown in lane 1 of each panel. Reaction mixtures corresponding to lane 2 of each panel contained all translation components except eIF1. Reference lanes A and G depict 18S rRNA sequence generated using the same primer. Complex I is arrested 21–24 nt from the 5′-end of β-globin mRNA. The 48S complex assembled on the AUG triplet is designated as “complex II (+15–17 nt from AUG).” The 48S complex assembled on the GUG triplet located 23 nt from the 5′-end of β-globin mRNA is designated as “+16 nt from GUG.”

    Journal: Genes & Development

    Article Title: Position of eukaryotic initiation factor eIF1 on the 40S ribosomal subunit determined by directed hydroxyl radical probing

    doi: 10.1101/gad.1141803

    Figure Lengend Snippet: Activities of eIF1 mutants in 48S complex formation. Toeprinting analysis of 48S complexes assembled on β-globin mRNA in the presence of 40S subunits, eIF2, eIF3, eIF4A, eIF4B, eIF4F, eIF1A, aminoacylated initiator tRNA, and the N-terminally truncated eIF1 Δ29 mutant ( A ) or Fe(II)-BABE-derivatized cysteineless and single-cysteine eIF1 mutants ( B ) as indicated. Reaction mixtures containing only β-globin mRNA are shown in lane 1 of each panel. Reaction mixtures corresponding to lane 2 of each panel contained all translation components except eIF1. Reference lanes A and G depict 18S rRNA sequence generated using the same primer. Complex I is arrested 21–24 nt from the 5′-end of β-globin mRNA. The 48S complex assembled on the AUG triplet is designated as “complex II (+15–17 nt from AUG).” The 48S complex assembled on the GUG triplet located 23 nt from the 5′-end of β-globin mRNA is designated as “+16 nt from GUG.”

    Article Snippet: For toeprinting analysis of 48S initiation complexes, reaction mixtures that contained 5 pmole of 40S subunits, 2μg of eIF2, 6 μg of eIF3, 0.5 μg of eIF4F, 2μg of eIF4A, 1 μg of eIF4B, 0.3 μg of eIF1A, 6 pmole of Met-tRNAi Met , 0.3 μg of native globin mRNA (Invitrogen), and 15 pmole of wild-type eIF1 or N-terminally truncated eIF1Δ29 mutant or 50 pmole of Fe(II)-BABE-derivatized eIF1 substitution mutants in 40 μL of buffer (20 mM Tris-HCl at pH 7.5, 100 mM KAc, 2.5 mM MgAc, 2 mM DTT, 1 mM ATP, 0.4 mM GTP, 0.25 mM spermidine) were incubated at 37°C for 10 min. 48S complex formation was analyzed by primer extension as described (Pestova et al. , ).

    Techniques: Mutagenesis, Sequencing, Generated

    Toeprint analysis of 48S ribosomal complex formation on β-globin mRNA with recombinant eIF4GI. The components of each reaction mixture are indicated above the lanes. Formation of complex II was quantified as described in Materials and Methods, and the value for eIF4F (lane 2) was set at 100.

    Journal: Molecular and Cellular Biology

    Article Title: Eukaryotic Translation Initiation Factor 4E (eIF4E) Binding Site and the Middle One-Third of eIF4GI Constitute the Core Domain for Cap-Dependent Translation, and the C-Terminal One-Third Functions as a Modulatory Region

    doi:

    Figure Lengend Snippet: Toeprint analysis of 48S ribosomal complex formation on β-globin mRNA with recombinant eIF4GI. The components of each reaction mixture are indicated above the lanes. Formation of complex II was quantified as described in Materials and Methods, and the value for eIF4F (lane 2) was set at 100.

    Article Snippet: Reaction mixtures (40 μl) containing native α- and β-globin mRNA (Life Technologies) (0.3 μg), His-eIF1 (0.5 μg), His-eIF1A (0.5 μg), eIF2 (3 μg), eIF3 (7 μg), FLAG-eIF4B (1 μg), Met-tRNAi Met (4 pmol), 40S ribosomal subunits (4 pmol), His- or FLAG-eIF4GI (2 μg, unless indicated), His-eIF4E (0.3 μg), and His-eIF4A (3 μg) were incubated in buffer (2 mM dithiothreitol, 100 mM KCl, 20 mM Tris-HCl [pH 7.6], 2.5 mM magnesium acetate, 100 U of RNasin [Promega], 1 mM ATP, 0.4 mM guanylyl imidodiphosphate [GMP-PNP], 250 μM spermidine) for 5 min at 30°C.

    Techniques: Recombinant

    Functional analysis of eIF4GI deletion mutants. (A) Schematic representation of eIF4GI deletion mutants. PABP, eIF4E, eIF4A, eIF3, and Mnk1 binding sites are indicated. (B) Toeprint analysis of 48S ribosomal complex formation on β-globin mRNA with eIF4GI deletion mutants. The reaction components are indicated above the lanes. The value for eIF4GI(157–1560) (lane 3) was set at 100. (C) Analysis of eIF4GI deletion mutants in a reticulocyte lysate translation system. Translation was performed as described in Materials and Methods. A rabbit reticulocyte lysate treated with rhinovirus 2A pro was supplemented with recombinant proteins as indicated and programmed for translation with the capped bicistronic mRNA CAT/EMCV IRES/LUC. For quantitation of luciferase (LUC) synthesis, the value obtained for translation in untreated lysate in the absence of additional proteins (lane 1) was set at 100. For the quantitation of CAT synthesis, the value obtained for translation in the treated lysate in the presence of eIF4E alone was subtracted as background, and then the value for treated lysate translated in the presence of eIF4E and eIF4GI(157–1560) (lane 4) was set at 100. (D) Western blotting of eIF4G deletion mutants. Recombinant protein preparations (∼1 μg) containing the same amount of eIF4GI according to Coomassie blue staining were subjected to SDS-PAGE (10% gel) and analyzed by Western blotting with anti-Xpress antibody (to detect the epitope located between the His tag and eIF4G coding sequence) or with anti-FLAG antibody.

    Journal: Molecular and Cellular Biology

    Article Title: Eukaryotic Translation Initiation Factor 4E (eIF4E) Binding Site and the Middle One-Third of eIF4GI Constitute the Core Domain for Cap-Dependent Translation, and the C-Terminal One-Third Functions as a Modulatory Region

    doi:

    Figure Lengend Snippet: Functional analysis of eIF4GI deletion mutants. (A) Schematic representation of eIF4GI deletion mutants. PABP, eIF4E, eIF4A, eIF3, and Mnk1 binding sites are indicated. (B) Toeprint analysis of 48S ribosomal complex formation on β-globin mRNA with eIF4GI deletion mutants. The reaction components are indicated above the lanes. The value for eIF4GI(157–1560) (lane 3) was set at 100. (C) Analysis of eIF4GI deletion mutants in a reticulocyte lysate translation system. Translation was performed as described in Materials and Methods. A rabbit reticulocyte lysate treated with rhinovirus 2A pro was supplemented with recombinant proteins as indicated and programmed for translation with the capped bicistronic mRNA CAT/EMCV IRES/LUC. For quantitation of luciferase (LUC) synthesis, the value obtained for translation in untreated lysate in the absence of additional proteins (lane 1) was set at 100. For the quantitation of CAT synthesis, the value obtained for translation in the treated lysate in the presence of eIF4E alone was subtracted as background, and then the value for treated lysate translated in the presence of eIF4E and eIF4GI(157–1560) (lane 4) was set at 100. (D) Western blotting of eIF4G deletion mutants. Recombinant protein preparations (∼1 μg) containing the same amount of eIF4GI according to Coomassie blue staining were subjected to SDS-PAGE (10% gel) and analyzed by Western blotting with anti-Xpress antibody (to detect the epitope located between the His tag and eIF4G coding sequence) or with anti-FLAG antibody.

    Article Snippet: Reaction mixtures (40 μl) containing native α- and β-globin mRNA (Life Technologies) (0.3 μg), His-eIF1 (0.5 μg), His-eIF1A (0.5 μg), eIF2 (3 μg), eIF3 (7 μg), FLAG-eIF4B (1 μg), Met-tRNAi Met (4 pmol), 40S ribosomal subunits (4 pmol), His- or FLAG-eIF4GI (2 μg, unless indicated), His-eIF4E (0.3 μg), and His-eIF4A (3 μg) were incubated in buffer (2 mM dithiothreitol, 100 mM KCl, 20 mM Tris-HCl [pH 7.6], 2.5 mM magnesium acetate, 100 U of RNasin [Promega], 1 mM ATP, 0.4 mM guanylyl imidodiphosphate [GMP-PNP], 250 μM spermidine) for 5 min at 30°C.

    Techniques: Functional Assay, Binding Assay, Recombinant, Quantitation Assay, Luciferase, Western Blot, Staining, SDS Page, Sequencing

    Elevation of γ-Globin Expression by Genome Editing Nucleases targeted to genomic sites create double-strand breaks (DSBs) that can be repaired by non-homologous end joining (NHEJ) or by homology-directed repair (HDR). Shown on the left are three examples for deletions causing increases in fetal hemoglobin production. The first shows the creation of a 13-kb deletion encompassing the δ- and β-globin gene. This deletion removes negative regulatory elements and positions the β-globin 3′ enhancer in close proximity to the γ-globin genes. The second example shows deletion of a repressor binding site in the γ-globin gene promoters. The third example shows creation of a frameshift in the coding region or a deletion of a positive DNA-regulatory element in one of the BLC11A erythroid-specific enhancers. Shown on the right are two examples for HDR-mediated correction of the sickle cell mutation. The first example shows an adeno-associated vector (AAV) that delivers the homologous arms (HAs), the therapeutic donor DNA, and a selectable marker (ITR: inverted terminal repeats). The second example shows a single stranded oligodeoxynucleotide containing 5′ and 3′ homologous arms (HAs) and the wild-type β-globin sequence. The diagram on the bottom right outlines the major steps involved in genome editing using autologous cell transplantation.

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Engineering Globin Gene Expression

    doi: 10.1016/j.omtm.2018.12.004

    Figure Lengend Snippet: Elevation of γ-Globin Expression by Genome Editing Nucleases targeted to genomic sites create double-strand breaks (DSBs) that can be repaired by non-homologous end joining (NHEJ) or by homology-directed repair (HDR). Shown on the left are three examples for deletions causing increases in fetal hemoglobin production. The first shows the creation of a 13-kb deletion encompassing the δ- and β-globin gene. This deletion removes negative regulatory elements and positions the β-globin 3′ enhancer in close proximity to the γ-globin genes. The second example shows deletion of a repressor binding site in the γ-globin gene promoters. The third example shows creation of a frameshift in the coding region or a deletion of a positive DNA-regulatory element in one of the BLC11A erythroid-specific enhancers. Shown on the right are two examples for HDR-mediated correction of the sickle cell mutation. The first example shows an adeno-associated vector (AAV) that delivers the homologous arms (HAs), the therapeutic donor DNA, and a selectable marker (ITR: inverted terminal repeats). The second example shows a single stranded oligodeoxynucleotide containing 5′ and 3′ homologous arms (HAs) and the wild-type β-globin sequence. The diagram on the bottom right outlines the major steps involved in genome editing using autologous cell transplantation.

    Article Snippet: In proof-of-concept studies, several laboratories demonstrated efficient HDR-mediated repair of the sickle cell mutation in human CD34+ or other hematopoietic stem and progenitor cells (HSPCs)., , Dever et al. used CRISPR/Cas9 to induce a DSB near the sickle cell mutation and an AAV vector to deliver a homologous DNA fragment that not only contained the wild-type β-globin gene sequence but also a selectable marker gene ( ).

    Techniques: Expressing, Non-Homologous End Joining, Binding Assay, Mutagenesis, Plasmid Preparation, Marker, Sequencing, Transplantation Assay

    Lentivirus Vector Employed in Globin Gene Therapy The diagram on top depicts the human β-globin gene locus, which consists of five genes (green boxes) that are expressed in a developmental stage-specific manner. High-level expression of the adult β-globin gene is mediated by the LCR HSs and the β-globin 3′ enhancer (blue ovals). Functional elements within all lentivirus vector systems include the long-terminal repeats (LTRs; gray boxes), splicing acceptor (SA) and donor (SD) sites, and the rev-responsive element (RRE), a structured RNA required for efficient viral replication. The TNS9 vector contains large segments of LCR elements HS2, HS3, and HS4, a wild-type β-globin gene, and a β-globin 3′enhancer. The HPV569 vector is similar to the TNS9 vector but contains a mutant β-globin gene that encodes a protein with a T to Q substitution at position 87. In addition, this vector contains two copies of the chicken HS4 (cHS4) insulator sequence in the LTRs. The Lenti-βAS3-FB vector is similar to HPV569 but expresses a β-globin protein with three amino acid substitutions (AS3) and contains single FB insulators in the LTRs. The GLOBE vector contains the β-globin gene and two large segments of the LCR (HS2 and HS3).

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Engineering Globin Gene Expression

    doi: 10.1016/j.omtm.2018.12.004

    Figure Lengend Snippet: Lentivirus Vector Employed in Globin Gene Therapy The diagram on top depicts the human β-globin gene locus, which consists of five genes (green boxes) that are expressed in a developmental stage-specific manner. High-level expression of the adult β-globin gene is mediated by the LCR HSs and the β-globin 3′ enhancer (blue ovals). Functional elements within all lentivirus vector systems include the long-terminal repeats (LTRs; gray boxes), splicing acceptor (SA) and donor (SD) sites, and the rev-responsive element (RRE), a structured RNA required for efficient viral replication. The TNS9 vector contains large segments of LCR elements HS2, HS3, and HS4, a wild-type β-globin gene, and a β-globin 3′enhancer. The HPV569 vector is similar to the TNS9 vector but contains a mutant β-globin gene that encodes a protein with a T to Q substitution at position 87. In addition, this vector contains two copies of the chicken HS4 (cHS4) insulator sequence in the LTRs. The Lenti-βAS3-FB vector is similar to HPV569 but expresses a β-globin protein with three amino acid substitutions (AS3) and contains single FB insulators in the LTRs. The GLOBE vector contains the β-globin gene and two large segments of the LCR (HS2 and HS3).

    Article Snippet: In proof-of-concept studies, several laboratories demonstrated efficient HDR-mediated repair of the sickle cell mutation in human CD34+ or other hematopoietic stem and progenitor cells (HSPCs)., , Dever et al. used CRISPR/Cas9 to induce a DSB near the sickle cell mutation and an AAV vector to deliver a homologous DNA fragment that not only contained the wild-type β-globin gene sequence but also a selectable marker gene ( ).

    Techniques: Plasmid Preparation, Expressing, Functional Assay, Mutagenesis, Sequencing

    Cleavage site preferences for falcipain-2 and falcipain-3. Preferences for amino acids (single letter code) at P 1 -P 4  and P 1 ′-P 4 ′ are shown schematically. At each position, the height of each amino acid corresponds to its frequency at cleavage sites based on analysis of α and β globin. WebLogo ( weblogo.berkeley.edu ), a free software for plotting consensus sequences, was used to plot P 4-  P 4 ′ preferences.

    Journal: PLoS ONE

    Article Title: Hemoglobin Cleavage Site-Specificity of the Plasmodium falciparum Cysteine Proteases Falcipain-2 and Falcipain-3

    doi: 10.1371/journal.pone.0005156

    Figure Lengend Snippet: Cleavage site preferences for falcipain-2 and falcipain-3. Preferences for amino acids (single letter code) at P 1 -P 4 and P 1 ′-P 4 ′ are shown schematically. At each position, the height of each amino acid corresponds to its frequency at cleavage sites based on analysis of α and β globin. WebLogo ( weblogo.berkeley.edu ), a free software for plotting consensus sequences, was used to plot P 4- P 4 ′ preferences.

    Article Snippet: Hydrolysis of globin peptides in the presence of H2 18 O Synthetic peptides of 12 amino acid length scanning the entire sequences of α and β globin, with 4 amino acid overlaps between peptides, were custom-ordered from Sigma-Genosys.

    Techniques: Software

    Hemoglobin cleavage sites for falcipain-2 and falcipain-3. A–D. Globin peptides and intact human hemoglobin. Cleavage sites for the two proteases based on analysis as described in Methods of hydrolysis of 12-mer peptides (top arrows, data from all time points) and intact hemoglobin (lower arrows, data from both 180 min and overnight time points) are shown for α and β globin. E. Intact human hemoglobin. The schematic shows α globin subunits in white and β globin subunits in yellow. P 1 residues at cleavage sites within helices are in red and within loops are in blue.

    Journal: PLoS ONE

    Article Title: Hemoglobin Cleavage Site-Specificity of the Plasmodium falciparum Cysteine Proteases Falcipain-2 and Falcipain-3

    doi: 10.1371/journal.pone.0005156

    Figure Lengend Snippet: Hemoglobin cleavage sites for falcipain-2 and falcipain-3. A–D. Globin peptides and intact human hemoglobin. Cleavage sites for the two proteases based on analysis as described in Methods of hydrolysis of 12-mer peptides (top arrows, data from all time points) and intact hemoglobin (lower arrows, data from both 180 min and overnight time points) are shown for α and β globin. E. Intact human hemoglobin. The schematic shows α globin subunits in white and β globin subunits in yellow. P 1 residues at cleavage sites within helices are in red and within loops are in blue.

    Article Snippet: Hydrolysis of globin peptides in the presence of H2 18 O Synthetic peptides of 12 amino acid length scanning the entire sequences of α and β globin, with 4 amino acid overlaps between peptides, were custom-ordered from Sigma-Genosys.

    Techniques:

    Time course for cleavage of globin peptides. Schematic representation of cleavage fragments over the indicated time points based on analysis of cleavage of 12-mer peptides spanning the sequences of α and β globin.

    Journal: PLoS ONE

    Article Title: Hemoglobin Cleavage Site-Specificity of the Plasmodium falciparum Cysteine Proteases Falcipain-2 and Falcipain-3

    doi: 10.1371/journal.pone.0005156

    Figure Lengend Snippet: Time course for cleavage of globin peptides. Schematic representation of cleavage fragments over the indicated time points based on analysis of cleavage of 12-mer peptides spanning the sequences of α and β globin.

    Article Snippet: Hydrolysis of globin peptides in the presence of H2 18 O Synthetic peptides of 12 amino acid length scanning the entire sequences of α and β globin, with 4 amino acid overlaps between peptides, were custom-ordered from Sigma-Genosys.

    Techniques:

    Yield of various truncated α-Hbs after purification. The different soluble fractions obtained after disruption of induced cells was applied on Glutathione Sepharose 4B in PBS (150 mM NaCl, 10 mM Na 2 HPO 4 , pH 7.4). After washing, various truncated GST-AHSP WT /GST-α-Hb complexes were eluted (elution buffer: 50 mM Tris-HCl, 20 mM reduced glutathione pH 8.0) and analyzed by (A) SDS-PAGE (12%) and (B) Western Blotting using anti-α globin antibodies.

    Journal: PLoS ONE

    Article Title: Role of α-Globin H Helix in the Building of Tetrameric Human Hemoglobin: Interaction with α-Hemoglobin Stabilizing Protein (AHSP) and Heme Molecule

    doi: 10.1371/journal.pone.0111395

    Figure Lengend Snippet: Yield of various truncated α-Hbs after purification. The different soluble fractions obtained after disruption of induced cells was applied on Glutathione Sepharose 4B in PBS (150 mM NaCl, 10 mM Na 2 HPO 4 , pH 7.4). After washing, various truncated GST-AHSP WT /GST-α-Hb complexes were eluted (elution buffer: 50 mM Tris-HCl, 20 mM reduced glutathione pH 8.0) and analyzed by (A) SDS-PAGE (12%) and (B) Western Blotting using anti-α globin antibodies.

    Article Snippet: Site-directed mutagenesis The different stop codons were introduced at the ad hoc position of α-globin by site-directed mutagenesis (Quick change Lightning Site Directed Mutagenesis kit, Stratagene, Agilent technologies, Santa Clara, CA, USA) using the pGEX6P-α-AHSP vector as template .

    Techniques: Purification, SDS Page, Western Blot

    H helix of human α-globin. (A) H helix sequence of the different truncated α-globins. The symbols indicate the amino-acids interacting with β-globin within the same αβ dimer (+) and those interacting with the heme molecule (•) [14] . The (x) symbol indicates the amino-acids interacting with AHSP. (B) Three-dimensional view of α-Hb. The representation was obtained from crystallographic data of oxyHb (2HHB) using the Pymol software. The truncated regions of the H helix investigated in this work are represented in blue (α-Hb1-138), purple (α-Hb1-134), red (α-Hb1-126), orange (α-Hb1-123) and grey (α-Hb1-117). The GH corner is shown in yellow.

    Journal: PLoS ONE

    Article Title: Role of α-Globin H Helix in the Building of Tetrameric Human Hemoglobin: Interaction with α-Hemoglobin Stabilizing Protein (AHSP) and Heme Molecule

    doi: 10.1371/journal.pone.0111395

    Figure Lengend Snippet: H helix of human α-globin. (A) H helix sequence of the different truncated α-globins. The symbols indicate the amino-acids interacting with β-globin within the same αβ dimer (+) and those interacting with the heme molecule (•) [14] . The (x) symbol indicates the amino-acids interacting with AHSP. (B) Three-dimensional view of α-Hb. The representation was obtained from crystallographic data of oxyHb (2HHB) using the Pymol software. The truncated regions of the H helix investigated in this work are represented in blue (α-Hb1-138), purple (α-Hb1-134), red (α-Hb1-126), orange (α-Hb1-123) and grey (α-Hb1-117). The GH corner is shown in yellow.

    Article Snippet: Site-directed mutagenesis The different stop codons were introduced at the ad hoc position of α-globin by site-directed mutagenesis (Quick change Lightning Site Directed Mutagenesis kit, Stratagene, Agilent technologies, Santa Clara, CA, USA) using the pGEX6P-α-AHSP vector as template .

    Techniques: Sequencing, Software

    Effect of truncation on the solubility of various truncated α-Hbs. The induced  E.coli  BL21(DE3) cells containing different pGEX-α-AHSP plasmids were disrupted. After centrifugation, soluble fractions were analyzed by (A) SDS-PAGE (12%) and by (B) Western Blotting using anti-α globin antibodies. The insoluble fractions were analyzed by (C) SDS-PAGE (12%) and by (D) Western Blotting using anti-α globin antibodies. Page ruler™ prestained protein ladder (Fermentas Thermo Fisher Scientific) was in the first lane.

    Journal: PLoS ONE

    Article Title: Role of α-Globin H Helix in the Building of Tetrameric Human Hemoglobin: Interaction with α-Hemoglobin Stabilizing Protein (AHSP) and Heme Molecule

    doi: 10.1371/journal.pone.0111395

    Figure Lengend Snippet: Effect of truncation on the solubility of various truncated α-Hbs. The induced E.coli BL21(DE3) cells containing different pGEX-α-AHSP plasmids were disrupted. After centrifugation, soluble fractions were analyzed by (A) SDS-PAGE (12%) and by (B) Western Blotting using anti-α globin antibodies. The insoluble fractions were analyzed by (C) SDS-PAGE (12%) and by (D) Western Blotting using anti-α globin antibodies. Page ruler™ prestained protein ladder (Fermentas Thermo Fisher Scientific) was in the first lane.

    Article Snippet: Site-directed mutagenesis The different stop codons were introduced at the ad hoc position of α-globin by site-directed mutagenesis (Quick change Lightning Site Directed Mutagenesis kit, Stratagene, Agilent technologies, Santa Clara, CA, USA) using the pGEX6P-α-AHSP vector as template .

    Techniques: Solubility, Centrifugation, SDS Page, Western Blot, Polyacrylamide Gel Electrophoresis

    Co-expression of truncated α-Hbs and AHSP WT  in  E.coli  BL21(DE3) cells. After 4 h induction by 0.2 mM IPTG, the  E. coli  cells containing the different pGEX-α-AHSP were lysed and total cell lysates were subjected to SDS-PAGE (12%) analysis (A) and Western Blotting using anti-α globin antibodies (B). All samples were prepared by boiling in SDS loading buffer. In all gels, GST-α-Hb WT  and GST-AHSP WT  are indicated by arrows. PAGE ruler™ prestained protein ladder (Fermentas Thermo Fisher Scientific, Waltham, MA, USA) was analyzed in the first lane.

    Journal: PLoS ONE

    Article Title: Role of α-Globin H Helix in the Building of Tetrameric Human Hemoglobin: Interaction with α-Hemoglobin Stabilizing Protein (AHSP) and Heme Molecule

    doi: 10.1371/journal.pone.0111395

    Figure Lengend Snippet: Co-expression of truncated α-Hbs and AHSP WT in E.coli BL21(DE3) cells. After 4 h induction by 0.2 mM IPTG, the E. coli cells containing the different pGEX-α-AHSP were lysed and total cell lysates were subjected to SDS-PAGE (12%) analysis (A) and Western Blotting using anti-α globin antibodies (B). All samples were prepared by boiling in SDS loading buffer. In all gels, GST-α-Hb WT and GST-AHSP WT are indicated by arrows. PAGE ruler™ prestained protein ladder (Fermentas Thermo Fisher Scientific, Waltham, MA, USA) was analyzed in the first lane.

    Article Snippet: Site-directed mutagenesis The different stop codons were introduced at the ad hoc position of α-globin by site-directed mutagenesis (Quick change Lightning Site Directed Mutagenesis kit, Stratagene, Agilent technologies, Santa Clara, CA, USA) using the pGEX6P-α-AHSP vector as template .

    Techniques: Expressing, SDS Page, Western Blot, Polyacrylamide Gel Electrophoresis

    Experimental Design and Analysis. ( a ) Blood RNA preparation steps from 6 human subjects: 3 controls ( c ) and 3 patients (P). RNA was used for preparing libraries with Ribo-Zero Gold (RZG) or Globin-Zero (GZ) kits as shown. In Experiment 1, a total of 33 libraries were prepared with either 250 ng total RNA for low (L) input or 900 ng for high (H) input. In Experiment 2 technical replicate libraries were prepared using 250 ng RNA from C1 and C2 with either RZG or Globin-Zero for a total of 12 libraries. Individual RNA-seq data files generated from the libraries shown in B and C were named according to the following convention: sample ID_RNA input_library method_replicate number, e.g., C1_L_GZ_1. ( b ) Fastq files were assessed for quality metrics, aligned and analyzed as shown. Correspondence between data analysis steps and results shown in Figs. 2 – 4 is indicated.

    Journal: Scientific Reports

    Article Title: RNA-Seq of human whole blood: Evaluation of globin RNA depletion on Ribo-Zero library method

    doi: 10.1038/s41598-020-62801-6

    Figure Lengend Snippet: Experimental Design and Analysis. ( a ) Blood RNA preparation steps from 6 human subjects: 3 controls ( c ) and 3 patients (P). RNA was used for preparing libraries with Ribo-Zero Gold (RZG) or Globin-Zero (GZ) kits as shown. In Experiment 1, a total of 33 libraries were prepared with either 250 ng total RNA for low (L) input or 900 ng for high (H) input. In Experiment 2 technical replicate libraries were prepared using 250 ng RNA from C1 and C2 with either RZG or Globin-Zero for a total of 12 libraries. Individual RNA-seq data files generated from the libraries shown in B and C were named according to the following convention: sample ID_RNA input_library method_replicate number, e.g., C1_L_GZ_1. ( b ) Fastq files were assessed for quality metrics, aligned and analyzed as shown. Correspondence between data analysis steps and results shown in Figs. 2 – 4 is indicated.

    Article Snippet: We tested two library preparation methods for the whole blood RNA samples: TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Gold and the TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Globin.

    Techniques: RNA Sequencing Assay, Generated

    Read distributions across methods and samples. ( a ) Estimated fraction of reads that map to hemoglobin (Hgb) genes, rRNA, and Other in method-paired datasets from 6 human donors (Experiment 1) and technical replicate datasets from C1 and C2 donors (Experiment 2) with either Globin-Zero (GZ) or Ribo-Zero-Gold (RZG). Proportions were estimated by mapping a subset of total trimmed reads to only Hgb gene sequences or only rRNA gene sequences, intentionally including multi-mapped reads. ( b ) HgbRNA levels in C1 and C2 libraries. Counts per million (CPM) expression levels measured in Experiment 2 GZ and RZG replicate libraries for Hgb genes were used to calculate the average log2 CPM for each gene in the C1 and C2 samples with each library method. Hgb gene family members with fewer than 20 raw counts in all libraries are not shown. ( c ) Ensembl gene biotype ( https://uswest.ensembl.org/info/genome/genebuild/biotypeshtml .) proportions from Experiment 2 utilizing the raw gene counts (number of uniquely-mapped reads that unambiguously map to only one gene) that were used for subsequent quantitative analysis. Technical replicates are averaged. RZG-Hg indicates gene counts from RZG libraries after bioinformatic removal of Hgb gene counts. ( d ) Gene biotype proportions of counts after TPM calculation which estimates the number of RNA transcripts from each gene by not only normalizing the raw gene counts by library depth as CPM does but also by accounting for gene length; this permits count comparison of differently-sized transcripts within a sample. Technical replicates are averaged.

    Journal: Scientific Reports

    Article Title: RNA-Seq of human whole blood: Evaluation of globin RNA depletion on Ribo-Zero library method

    doi: 10.1038/s41598-020-62801-6

    Figure Lengend Snippet: Read distributions across methods and samples. ( a ) Estimated fraction of reads that map to hemoglobin (Hgb) genes, rRNA, and Other in method-paired datasets from 6 human donors (Experiment 1) and technical replicate datasets from C1 and C2 donors (Experiment 2) with either Globin-Zero (GZ) or Ribo-Zero-Gold (RZG). Proportions were estimated by mapping a subset of total trimmed reads to only Hgb gene sequences or only rRNA gene sequences, intentionally including multi-mapped reads. ( b ) HgbRNA levels in C1 and C2 libraries. Counts per million (CPM) expression levels measured in Experiment 2 GZ and RZG replicate libraries for Hgb genes were used to calculate the average log2 CPM for each gene in the C1 and C2 samples with each library method. Hgb gene family members with fewer than 20 raw counts in all libraries are not shown. ( c ) Ensembl gene biotype ( https://uswest.ensembl.org/info/genome/genebuild/biotypeshtml .) proportions from Experiment 2 utilizing the raw gene counts (number of uniquely-mapped reads that unambiguously map to only one gene) that were used for subsequent quantitative analysis. Technical replicates are averaged. RZG-Hg indicates gene counts from RZG libraries after bioinformatic removal of Hgb gene counts. ( d ) Gene biotype proportions of counts after TPM calculation which estimates the number of RNA transcripts from each gene by not only normalizing the raw gene counts by library depth as CPM does but also by accounting for gene length; this permits count comparison of differently-sized transcripts within a sample. Technical replicates are averaged.

    Article Snippet: We tested two library preparation methods for the whole blood RNA samples: TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Gold and the TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Globin.

    Techniques: Expressing

    Human β-globin reporter gene. ( A ) Detail of the reporter construct (See ‘Materials and methods’). Top schematic drawn to scale. ( B ) Imaging of gene expression from nascent transcripts (arrow) to protein product (blue channel shows CFP-SKL protein product of the reporter gene accumulating in peroxisomes). Images are maximum intensity projections of z-stacks (Δz = 0.25 μm, exposure = 1 s). ( C ) Splicing efficiency and ( D ) poly(A) tail site/length of the β-globin reporter, measured in three conditions: mock-transfected cells and cells transfected with either wild type (WT) or mutant (S34F) splicing factor U2AF1 (see Materials and methods). Error bars are SEM calculated over four measurements. ( E ) Expected fluorescence time profiles for a single transcript. When the PP7 cassette is transcribed, the red fluorescence signal increases progressively (as RNA stem loops are formed) and plateaus once the polymerase exits the cassette. The same applies to the green fluorescence signal when the PP7 cassette is transcribed. If splicing is post-release, red and green signals drop simultaneously when the unspliced RNA is released and diffuse away. If splicing is pre-release, the red fluorescence drops before the green fluorescence reflecting that intron removal occurred before the release of a spliced transcript. DOI: http://dx.doi.org/10.7554/eLife.03939.004

    Journal: eLife

    Article Title: Kinetic competition during the transcription cycle results in stochastic RNA processing

    doi: 10.7554/eLife.03939

    Figure Lengend Snippet: Human β-globin reporter gene. ( A ) Detail of the reporter construct (See ‘Materials and methods’). Top schematic drawn to scale. ( B ) Imaging of gene expression from nascent transcripts (arrow) to protein product (blue channel shows CFP-SKL protein product of the reporter gene accumulating in peroxisomes). Images are maximum intensity projections of z-stacks (Δz = 0.25 μm, exposure = 1 s). ( C ) Splicing efficiency and ( D ) poly(A) tail site/length of the β-globin reporter, measured in three conditions: mock-transfected cells and cells transfected with either wild type (WT) or mutant (S34F) splicing factor U2AF1 (see Materials and methods). Error bars are SEM calculated over four measurements. ( E ) Expected fluorescence time profiles for a single transcript. When the PP7 cassette is transcribed, the red fluorescence signal increases progressively (as RNA stem loops are formed) and plateaus once the polymerase exits the cassette. The same applies to the green fluorescence signal when the PP7 cassette is transcribed. If splicing is post-release, red and green signals drop simultaneously when the unspliced RNA is released and diffuse away. If splicing is pre-release, the red fluorescence drops before the green fluorescence reflecting that intron removal occurred before the release of a spliced transcript. DOI: http://dx.doi.org/10.7554/eLife.03939.004

    Article Snippet: The reads were trimmed to remove low quality sequences (Trimmomatic software) and were aligned to both the human genome (hg19) and the sequence of the β-globin reporter gene plasmid (Bowtie2 and Illumina CASAVA Eland softwares).

    Techniques: Construct, Imaging, Expressing, Transfection, Mutagenesis, Fluorescence

    Schematic of β-globin transcription cycle kinetics. Transcript synthesis and processing can occur through different pathways, the choice of which is governed by a kinetic competition between transcription and splicing. After transcription of the 3′ splice site, intron removal takes about 260 s and elongation until the end of the gene, about 55 s. Hence, splicing does not occur during elongation. The transcript is retained at the 3′end of the gene for a stochastic amount of time that can be shorter or longer than the remaining time to excise the intron. This results in two possible outcomes: either an unspliced pre-mRNA is released and then spliced very rapidly or splicing occurs while the transcript is retained on chromatin before being released. DOI: http://dx.doi.org/10.7554/eLife.03939.025

    Journal: eLife

    Article Title: Kinetic competition during the transcription cycle results in stochastic RNA processing

    doi: 10.7554/eLife.03939

    Figure Lengend Snippet: Schematic of β-globin transcription cycle kinetics. Transcript synthesis and processing can occur through different pathways, the choice of which is governed by a kinetic competition between transcription and splicing. After transcription of the 3′ splice site, intron removal takes about 260 s and elongation until the end of the gene, about 55 s. Hence, splicing does not occur during elongation. The transcript is retained at the 3′end of the gene for a stochastic amount of time that can be shorter or longer than the remaining time to excise the intron. This results in two possible outcomes: either an unspliced pre-mRNA is released and then spliced very rapidly or splicing occurs while the transcript is retained on chromatin before being released. DOI: http://dx.doi.org/10.7554/eLife.03939.025

    Article Snippet: The reads were trimmed to remove low quality sequences (Trimmomatic software) and were aligned to both the human genome (hg19) and the sequence of the β-globin reporter gene plasmid (Bowtie2 and Illumina CASAVA Eland softwares).

    Techniques:

    Integration site of the β-globin reporter and copy number analysis. ( A ) Example of an integration site of the reporter plasmid as identified by whole genome sequencing. Reads aligning to both genomic and plasmid sequences are shown at the bottom. The alignments identify the genomic position of the insertion and the location of the breaks in the plasmid. The number of repeats of the plasmid at the insertion site cannot be known from sequencing data. ( B ) Three insertion sites were identified in the cell line. ( C ) Semi-quantitative PCR was used to confirm the insertion sites and to estimate the total copy number of the integrated plasmid construct in the cell line. ( D ) Quantification of the PCR products shows, as expected, that amplicons internal to the plasmid were more amplified than the amplicons at the junctions. ( E ) Calibration curves were made by amplifying varying amounts of G-block DNA carrying the same primer pairs as used in ( C ). ( F ) Correcting the data in ( D ) with the calibration curves in ( E ) and taking the ratio of internal-to-junction PCR products yields a total copy number of 5.48 ± 1.47. Error is SEM. DOI: http://dx.doi.org/10.7554/eLife.03939.005

    Journal: eLife

    Article Title: Kinetic competition during the transcription cycle results in stochastic RNA processing

    doi: 10.7554/eLife.03939

    Figure Lengend Snippet: Integration site of the β-globin reporter and copy number analysis. ( A ) Example of an integration site of the reporter plasmid as identified by whole genome sequencing. Reads aligning to both genomic and plasmid sequences are shown at the bottom. The alignments identify the genomic position of the insertion and the location of the breaks in the plasmid. The number of repeats of the plasmid at the insertion site cannot be known from sequencing data. ( B ) Three insertion sites were identified in the cell line. ( C ) Semi-quantitative PCR was used to confirm the insertion sites and to estimate the total copy number of the integrated plasmid construct in the cell line. ( D ) Quantification of the PCR products shows, as expected, that amplicons internal to the plasmid were more amplified than the amplicons at the junctions. ( E ) Calibration curves were made by amplifying varying amounts of G-block DNA carrying the same primer pairs as used in ( C ). ( F ) Correcting the data in ( D ) with the calibration curves in ( E ) and taking the ratio of internal-to-junction PCR products yields a total copy number of 5.48 ± 1.47. Error is SEM. DOI: http://dx.doi.org/10.7554/eLife.03939.005

    Article Snippet: The reads were trimmed to remove low quality sequences (Trimmomatic software) and were aligned to both the human genome (hg19) and the sequence of the β-globin reporter gene plasmid (Bowtie2 and Illumina CASAVA Eland softwares).

    Techniques: Plasmid Preparation, Sequencing, Real-time Polymerase Chain Reaction, Construct, Polymerase Chain Reaction, Amplification, Blocking Assay