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  • 90
    Thermo Fisher tbe gels
    Digestion with micrococcal nuclease yields a robust ribosome profiling assay. ( A ) Digestion of polysomes with RNase I degrades ribosomes. A lysate was made from S2 cells using a previous version of our protocol. Aliquots of this lysate were digested with increasing amounts of RNase I, and resolved on 10–50% sucrose gradients. As amounts of RNase I increase, the heights of all peaks—including the monosomal (80S) peak—decrease before polysomes are fully resolved to monosomes. ( B ) as in ( A ), but using micrococcal nuclease (MNase) and our current protocol. From 0.5 to 2 U MNase/μg total RNA, monosomes are resolved with no reduction in the size of the monosome peak. This result indicates that Drosophila ribosomes are stable to MNase over a broad range of concentrations, whereas the mRNA between ribosomes is digested. ( C ) Ribosome protection assay. A 320 nucleotide fragment of enolase (FlyBase accession: FBgn0000579) was amplified using oligos oJGD123 oJGD124 ( Supplementary file 2 ). A body-labeled probe against this sequence was transcribed from this template using α32P-UTP and the T7 MaxiScript kit (Ambion). S2 cell lysates were prepared as in methods and aliquoted. Aliquots were digested as in methods, except with 0, 0.5, 1, 2, 3 or 4 U MNase/μg total RNA. Monosomes were sedimented through a sucrose cushion, <t>resuspended</t> in 600 μl 10 mM Tris pH 7.0, and their RNAs extracted as in ‘Materials and methods’. Concentrations were determined using a NanoDrop spectrophotometer. 5 μg of each sample was hybridized to 50,000 CPM of probe overnight at 42°C. Single-stranded regions were digested with RNase A/T1 and the remaining footprint: probe duplexes detected using the mirVana micro-RNA detection kit (Ambion), resolved on a 15% <t>TBE-urea</t> gel (Invitrogen), and visualized on a Storm phosphorimager (Molecular Dynamics by GE Healthcare Bio-Sciences, Pittsburgh, PA). For size markers, we end-labeled the Novex 10 bp dsDNA ladder (Invitrogen) with 32P. Over two-fold range of nuclease concentrations, the ∼30 nt peak corresponding to ribosome-protected footprints remains constant in size and intensity, indicating a lack of degradation consistent with the unchanged monosome peak height across this range of digestion conditions in ( B ). Also visible is a roughly 60 nt band which we infer to be protected by adjacent ribosomes (disomes) that sterically exclude the nuclease. This interpretation is consistent with the presence of a small disome peak in digested samples (c.f. panels B and D , and Figure 1A ). ( D ) A polysome lysate was prepared from S2 cells and resolved in 10–50% sucrose gradients, with or without prior digestion with 3 U MNase/μg total RNA ( E ) A culture of S2 cells was split into aliquots and processed using our current protocol as if they were independent samples. Total counts aligning to the coding region of each gene were tabulated in each replicate. Genes sharing at least 128 footprint counts between replicates (red) are well-correlated, demonstrating the assay is robust (see full discussion in Figure 1—figure supplement 2 ). Source data may be found in supplementary table 1 (at Dryad: Dunn et al., 2013 ). DOI: http://dx.doi.org/10.7554/eLife.01179.004
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    89
    Thermo Fisher novex tbe gel
    Digestion with micrococcal nuclease yields a robust ribosome profiling assay. ( A ) Digestion of polysomes with RNase I degrades ribosomes. A lysate was made from S2 cells using a previous version of our protocol. Aliquots of this lysate were digested with increasing amounts of RNase I, and resolved on 10–50% sucrose gradients. As amounts of RNase I increase, the heights of all peaks—including the monosomal (80S) peak—decrease before polysomes are fully resolved to monosomes. ( B ) as in ( A ), but using micrococcal nuclease (MNase) and our current protocol. From 0.5 to 2 U MNase/μg total RNA, monosomes are resolved with no reduction in the size of the monosome peak. This result indicates that Drosophila ribosomes are stable to MNase over a broad range of concentrations, whereas the mRNA between ribosomes is digested. ( C ) Ribosome protection assay. A 320 nucleotide fragment of enolase (FlyBase accession: FBgn0000579) was amplified using oligos oJGD123 oJGD124 ( Supplementary file 2 ). A body-labeled probe against this sequence was transcribed from this template using α32P-UTP and the T7 MaxiScript kit (Ambion). S2 cell lysates were prepared as in methods and aliquoted. Aliquots were digested as in methods, except with 0, 0.5, 1, 2, 3 or 4 U MNase/μg total RNA. Monosomes were sedimented through a sucrose cushion, <t>resuspended</t> in 600 μl 10 mM Tris pH 7.0, and their RNAs extracted as in ‘Materials and methods’. Concentrations were determined using a NanoDrop spectrophotometer. 5 μg of each sample was hybridized to 50,000 CPM of probe overnight at 42°C. Single-stranded regions were digested with RNase A/T1 and the remaining footprint: probe duplexes detected using the mirVana micro-RNA detection kit (Ambion), resolved on a 15% <t>TBE-urea</t> gel (Invitrogen), and visualized on a Storm phosphorimager (Molecular Dynamics by GE Healthcare Bio-Sciences, Pittsburgh, PA). For size markers, we end-labeled the Novex 10 bp dsDNA ladder (Invitrogen) with 32P. Over two-fold range of nuclease concentrations, the ∼30 nt peak corresponding to ribosome-protected footprints remains constant in size and intensity, indicating a lack of degradation consistent with the unchanged monosome peak height across this range of digestion conditions in ( B ). Also visible is a roughly 60 nt band which we infer to be protected by adjacent ribosomes (disomes) that sterically exclude the nuclease. This interpretation is consistent with the presence of a small disome peak in digested samples (c.f. panels B and D , and Figure 1A ). ( D ) A polysome lysate was prepared from S2 cells and resolved in 10–50% sucrose gradients, with or without prior digestion with 3 U MNase/μg total RNA ( E ) A culture of S2 cells was split into aliquots and processed using our current protocol as if they were independent samples. Total counts aligning to the coding region of each gene were tabulated in each replicate. Genes sharing at least 128 footprint counts between replicates (red) are well-correlated, demonstrating the assay is robust (see full discussion in Figure 1—figure supplement 2 ). Source data may be found in supplementary table 1 (at Dryad: Dunn et al., 2013 ). DOI: http://dx.doi.org/10.7554/eLife.01179.004
    Novex Tbe Gel, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 359 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Bio-Rad tbe gels
    Digestion with micrococcal nuclease yields a robust ribosome profiling assay. ( A ) Digestion of polysomes with RNase I degrades ribosomes. A lysate was made from S2 cells using a previous version of our protocol. Aliquots of this lysate were digested with increasing amounts of RNase I, and resolved on 10–50% sucrose gradients. As amounts of RNase I increase, the heights of all peaks—including the monosomal (80S) peak—decrease before polysomes are fully resolved to monosomes. ( B ) as in ( A ), but using micrococcal nuclease (MNase) and our current protocol. From 0.5 to 2 U MNase/μg total RNA, monosomes are resolved with no reduction in the size of the monosome peak. This result indicates that Drosophila ribosomes are stable to MNase over a broad range of concentrations, whereas the mRNA between ribosomes is digested. ( C ) Ribosome protection assay. A 320 nucleotide fragment of enolase (FlyBase accession: FBgn0000579) was amplified using oligos oJGD123 oJGD124 ( Supplementary file 2 ). A body-labeled probe against this sequence was transcribed from this template using α32P-UTP and the T7 MaxiScript kit (Ambion). S2 cell lysates were prepared as in methods and aliquoted. Aliquots were digested as in methods, except with 0, 0.5, 1, 2, 3 or 4 U MNase/μg total RNA. Monosomes were sedimented through a sucrose cushion, <t>resuspended</t> in 600 μl 10 mM Tris pH 7.0, and their RNAs extracted as in ‘Materials and methods’. Concentrations were determined using a NanoDrop spectrophotometer. 5 μg of each sample was hybridized to 50,000 CPM of probe overnight at 42°C. Single-stranded regions were digested with RNase A/T1 and the remaining footprint: probe duplexes detected using the mirVana micro-RNA detection kit (Ambion), resolved on a 15% <t>TBE-urea</t> gel (Invitrogen), and visualized on a Storm phosphorimager (Molecular Dynamics by GE Healthcare Bio-Sciences, Pittsburgh, PA). For size markers, we end-labeled the Novex 10 bp dsDNA ladder (Invitrogen) with 32P. Over two-fold range of nuclease concentrations, the ∼30 nt peak corresponding to ribosome-protected footprints remains constant in size and intensity, indicating a lack of degradation consistent with the unchanged monosome peak height across this range of digestion conditions in ( B ). Also visible is a roughly 60 nt band which we infer to be protected by adjacent ribosomes (disomes) that sterically exclude the nuclease. This interpretation is consistent with the presence of a small disome peak in digested samples (c.f. panels B and D , and Figure 1A ). ( D ) A polysome lysate was prepared from S2 cells and resolved in 10–50% sucrose gradients, with or without prior digestion with 3 U MNase/μg total RNA ( E ) A culture of S2 cells was split into aliquots and processed using our current protocol as if they were independent samples. Total counts aligning to the coding region of each gene were tabulated in each replicate. Genes sharing at least 128 footprint counts between replicates (red) are well-correlated, demonstrating the assay is robust (see full discussion in Figure 1—figure supplement 2 ). Source data may be found in supplementary table 1 (at Dryad: Dunn et al., 2013 ). DOI: http://dx.doi.org/10.7554/eLife.01179.004
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    91
    Lonza tbe gels
    Digestion with micrococcal nuclease yields a robust ribosome profiling assay. ( A ) Digestion of polysomes with RNase I degrades ribosomes. A lysate was made from S2 cells using a previous version of our protocol. Aliquots of this lysate were digested with increasing amounts of RNase I, and resolved on 10–50% sucrose gradients. As amounts of RNase I increase, the heights of all peaks—including the monosomal (80S) peak—decrease before polysomes are fully resolved to monosomes. ( B ) as in ( A ), but using micrococcal nuclease (MNase) and our current protocol. From 0.5 to 2 U MNase/μg total RNA, monosomes are resolved with no reduction in the size of the monosome peak. This result indicates that Drosophila ribosomes are stable to MNase over a broad range of concentrations, whereas the mRNA between ribosomes is digested. ( C ) Ribosome protection assay. A 320 nucleotide fragment of enolase (FlyBase accession: FBgn0000579) was amplified using oligos oJGD123 oJGD124 ( Supplementary file 2 ). A body-labeled probe against this sequence was transcribed from this template using α32P-UTP and the T7 MaxiScript kit (Ambion). S2 cell lysates were prepared as in methods and aliquoted. Aliquots were digested as in methods, except with 0, 0.5, 1, 2, 3 or 4 U MNase/μg total RNA. Monosomes were sedimented through a sucrose cushion, <t>resuspended</t> in 600 μl 10 mM Tris pH 7.0, and their RNAs extracted as in ‘Materials and methods’. Concentrations were determined using a NanoDrop spectrophotometer. 5 μg of each sample was hybridized to 50,000 CPM of probe overnight at 42°C. Single-stranded regions were digested with RNase A/T1 and the remaining footprint: probe duplexes detected using the mirVana micro-RNA detection kit (Ambion), resolved on a 15% <t>TBE-urea</t> gel (Invitrogen), and visualized on a Storm phosphorimager (Molecular Dynamics by GE Healthcare Bio-Sciences, Pittsburgh, PA). For size markers, we end-labeled the Novex 10 bp dsDNA ladder (Invitrogen) with 32P. Over two-fold range of nuclease concentrations, the ∼30 nt peak corresponding to ribosome-protected footprints remains constant in size and intensity, indicating a lack of degradation consistent with the unchanged monosome peak height across this range of digestion conditions in ( B ). Also visible is a roughly 60 nt band which we infer to be protected by adjacent ribosomes (disomes) that sterically exclude the nuclease. This interpretation is consistent with the presence of a small disome peak in digested samples (c.f. panels B and D , and Figure 1A ). ( D ) A polysome lysate was prepared from S2 cells and resolved in 10–50% sucrose gradients, with or without prior digestion with 3 U MNase/μg total RNA ( E ) A culture of S2 cells was split into aliquots and processed using our current protocol as if they were independent samples. Total counts aligning to the coding region of each gene were tabulated in each replicate. Genes sharing at least 128 footprint counts between replicates (red) are well-correlated, demonstrating the assay is robust (see full discussion in Figure 1—figure supplement 2 ). Source data may be found in supplementary table 1 (at Dryad: Dunn et al., 2013 ). DOI: http://dx.doi.org/10.7554/eLife.01179.004
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    99
    Thermo Fisher novex tbe page gels
    Digestion with micrococcal nuclease yields a robust ribosome profiling assay. ( A ) Digestion of polysomes with RNase I degrades ribosomes. A lysate was made from S2 cells using a previous version of our protocol. Aliquots of this lysate were digested with increasing amounts of RNase I, and resolved on 10–50% sucrose gradients. As amounts of RNase I increase, the heights of all peaks—including the monosomal (80S) peak—decrease before polysomes are fully resolved to monosomes. ( B ) as in ( A ), but using micrococcal nuclease (MNase) and our current protocol. From 0.5 to 2 U MNase/μg total RNA, monosomes are resolved with no reduction in the size of the monosome peak. This result indicates that Drosophila ribosomes are stable to MNase over a broad range of concentrations, whereas the mRNA between ribosomes is digested. ( C ) Ribosome protection assay. A 320 nucleotide fragment of enolase (FlyBase accession: FBgn0000579) was amplified using oligos oJGD123 oJGD124 ( Supplementary file 2 ). A body-labeled probe against this sequence was transcribed from this template using α32P-UTP and the T7 MaxiScript kit (Ambion). S2 cell lysates were prepared as in methods and aliquoted. Aliquots were digested as in methods, except with 0, 0.5, 1, 2, 3 or 4 U MNase/μg total RNA. Monosomes were sedimented through a sucrose cushion, <t>resuspended</t> in 600 μl 10 mM Tris pH 7.0, and their RNAs extracted as in ‘Materials and methods’. Concentrations were determined using a NanoDrop spectrophotometer. 5 μg of each sample was hybridized to 50,000 CPM of probe overnight at 42°C. Single-stranded regions were digested with RNase A/T1 and the remaining footprint: probe duplexes detected using the mirVana micro-RNA detection kit (Ambion), resolved on a 15% <t>TBE-urea</t> gel (Invitrogen), and visualized on a Storm phosphorimager (Molecular Dynamics by GE Healthcare Bio-Sciences, Pittsburgh, PA). For size markers, we end-labeled the Novex 10 bp dsDNA ladder (Invitrogen) with 32P. Over two-fold range of nuclease concentrations, the ∼30 nt peak corresponding to ribosome-protected footprints remains constant in size and intensity, indicating a lack of degradation consistent with the unchanged monosome peak height across this range of digestion conditions in ( B ). Also visible is a roughly 60 nt band which we infer to be protected by adjacent ribosomes (disomes) that sterically exclude the nuclease. This interpretation is consistent with the presence of a small disome peak in digested samples (c.f. panels B and D , and Figure 1A ). ( D ) A polysome lysate was prepared from S2 cells and resolved in 10–50% sucrose gradients, with or without prior digestion with 3 U MNase/μg total RNA ( E ) A culture of S2 cells was split into aliquots and processed using our current protocol as if they were independent samples. Total counts aligning to the coding region of each gene were tabulated in each replicate. Genes sharing at least 128 footprint counts between replicates (red) are well-correlated, demonstrating the assay is robust (see full discussion in Figure 1—figure supplement 2 ). Source data may be found in supplementary table 1 (at Dryad: Dunn et al., 2013 ). DOI: http://dx.doi.org/10.7554/eLife.01179.004
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    99
    Bio-Rad ready gel tbe gel
    Digestion with micrococcal nuclease yields a robust ribosome profiling assay. ( A ) Digestion of polysomes with RNase I degrades ribosomes. A lysate was made from S2 cells using a previous version of our protocol. Aliquots of this lysate were digested with increasing amounts of RNase I, and resolved on 10–50% sucrose gradients. As amounts of RNase I increase, the heights of all peaks—including the monosomal (80S) peak—decrease before polysomes are fully resolved to monosomes. ( B ) as in ( A ), but using micrococcal nuclease (MNase) and our current protocol. From 0.5 to 2 U MNase/μg total RNA, monosomes are resolved with no reduction in the size of the monosome peak. This result indicates that Drosophila ribosomes are stable to MNase over a broad range of concentrations, whereas the mRNA between ribosomes is digested. ( C ) Ribosome protection assay. A 320 nucleotide fragment of enolase (FlyBase accession: FBgn0000579) was amplified using oligos oJGD123 oJGD124 ( Supplementary file 2 ). A body-labeled probe against this sequence was transcribed from this template using α32P-UTP and the T7 MaxiScript kit (Ambion). S2 cell lysates were prepared as in methods and aliquoted. Aliquots were digested as in methods, except with 0, 0.5, 1, 2, 3 or 4 U MNase/μg total RNA. Monosomes were sedimented through a sucrose cushion, <t>resuspended</t> in 600 μl 10 mM Tris pH 7.0, and their RNAs extracted as in ‘Materials and methods’. Concentrations were determined using a NanoDrop spectrophotometer. 5 μg of each sample was hybridized to 50,000 CPM of probe overnight at 42°C. Single-stranded regions were digested with RNase A/T1 and the remaining footprint: probe duplexes detected using the mirVana micro-RNA detection kit (Ambion), resolved on a 15% <t>TBE-urea</t> gel (Invitrogen), and visualized on a Storm phosphorimager (Molecular Dynamics by GE Healthcare Bio-Sciences, Pittsburgh, PA). For size markers, we end-labeled the Novex 10 bp dsDNA ladder (Invitrogen) with 32P. Over two-fold range of nuclease concentrations, the ∼30 nt peak corresponding to ribosome-protected footprints remains constant in size and intensity, indicating a lack of degradation consistent with the unchanged monosome peak height across this range of digestion conditions in ( B ). Also visible is a roughly 60 nt band which we infer to be protected by adjacent ribosomes (disomes) that sterically exclude the nuclease. This interpretation is consistent with the presence of a small disome peak in digested samples (c.f. panels B and D , and Figure 1A ). ( D ) A polysome lysate was prepared from S2 cells and resolved in 10–50% sucrose gradients, with or without prior digestion with 3 U MNase/μg total RNA ( E ) A culture of S2 cells was split into aliquots and processed using our current protocol as if they were independent samples. Total counts aligning to the coding region of each gene were tabulated in each replicate. Genes sharing at least 128 footprint counts between replicates (red) are well-correlated, demonstrating the assay is robust (see full discussion in Figure 1—figure supplement 2 ). Source data may be found in supplementary table 1 (at Dryad: Dunn et al., 2013 ). DOI: http://dx.doi.org/10.7554/eLife.01179.004
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    tbe  (Bio-Rad)
    95
    Bio-Rad tbe
    Digestion with micrococcal nuclease yields a robust ribosome profiling assay. ( A ) Digestion of polysomes with RNase I degrades ribosomes. A lysate was made from S2 cells using a previous version of our protocol. Aliquots of this lysate were digested with increasing amounts of RNase I, and resolved on 10–50% sucrose gradients. As amounts of RNase I increase, the heights of all peaks—including the monosomal (80S) peak—decrease before polysomes are fully resolved to monosomes. ( B ) as in ( A ), but using micrococcal nuclease (MNase) and our current protocol. From 0.5 to 2 U MNase/μg total RNA, monosomes are resolved with no reduction in the size of the monosome peak. This result indicates that Drosophila ribosomes are stable to MNase over a broad range of concentrations, whereas the mRNA between ribosomes is digested. ( C ) Ribosome protection assay. A 320 nucleotide fragment of enolase (FlyBase accession: FBgn0000579) was amplified using oligos oJGD123 oJGD124 ( Supplementary file 2 ). A body-labeled probe against this sequence was transcribed from this template using α32P-UTP and the T7 MaxiScript kit (Ambion). S2 cell lysates were prepared as in methods and aliquoted. Aliquots were digested as in methods, except with 0, 0.5, 1, 2, 3 or 4 U MNase/μg total RNA. Monosomes were sedimented through a sucrose cushion, <t>resuspended</t> in 600 μl 10 mM Tris pH 7.0, and their RNAs extracted as in ‘Materials and methods’. Concentrations were determined using a NanoDrop spectrophotometer. 5 μg of each sample was hybridized to 50,000 CPM of probe overnight at 42°C. Single-stranded regions were digested with RNase A/T1 and the remaining footprint: probe duplexes detected using the mirVana micro-RNA detection kit (Ambion), resolved on a 15% <t>TBE-urea</t> gel (Invitrogen), and visualized on a Storm phosphorimager (Molecular Dynamics by GE Healthcare Bio-Sciences, Pittsburgh, PA). For size markers, we end-labeled the Novex 10 bp dsDNA ladder (Invitrogen) with 32P. Over two-fold range of nuclease concentrations, the ∼30 nt peak corresponding to ribosome-protected footprints remains constant in size and intensity, indicating a lack of degradation consistent with the unchanged monosome peak height across this range of digestion conditions in ( B ). Also visible is a roughly 60 nt band which we infer to be protected by adjacent ribosomes (disomes) that sterically exclude the nuclease. This interpretation is consistent with the presence of a small disome peak in digested samples (c.f. panels B and D , and Figure 1A ). ( D ) A polysome lysate was prepared from S2 cells and resolved in 10–50% sucrose gradients, with or without prior digestion with 3 U MNase/μg total RNA ( E ) A culture of S2 cells was split into aliquots and processed using our current protocol as if they were independent samples. Total counts aligning to the coding region of each gene were tabulated in each replicate. Genes sharing at least 128 footprint counts between replicates (red) are well-correlated, demonstrating the assay is robust (see full discussion in Figure 1—figure supplement 2 ). Source data may be found in supplementary table 1 (at Dryad: Dunn et al., 2013 ). DOI: http://dx.doi.org/10.7554/eLife.01179.004
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    99
    Thermo Fisher novex precast tbe gels
    Digestion with micrococcal nuclease yields a robust ribosome profiling assay. ( A ) Digestion of polysomes with RNase I degrades ribosomes. A lysate was made from S2 cells using a previous version of our protocol. Aliquots of this lysate were digested with increasing amounts of RNase I, and resolved on 10–50% sucrose gradients. As amounts of RNase I increase, the heights of all peaks—including the monosomal (80S) peak—decrease before polysomes are fully resolved to monosomes. ( B ) as in ( A ), but using micrococcal nuclease (MNase) and our current protocol. From 0.5 to 2 U MNase/μg total RNA, monosomes are resolved with no reduction in the size of the monosome peak. This result indicates that Drosophila ribosomes are stable to MNase over a broad range of concentrations, whereas the mRNA between ribosomes is digested. ( C ) Ribosome protection assay. A 320 nucleotide fragment of enolase (FlyBase accession: FBgn0000579) was amplified using oligos oJGD123 oJGD124 ( Supplementary file 2 ). A body-labeled probe against this sequence was transcribed from this template using α32P-UTP and the T7 MaxiScript kit (Ambion). S2 cell lysates were prepared as in methods and aliquoted. Aliquots were digested as in methods, except with 0, 0.5, 1, 2, 3 or 4 U MNase/μg total RNA. Monosomes were sedimented through a sucrose cushion, <t>resuspended</t> in 600 μl 10 mM Tris pH 7.0, and their RNAs extracted as in ‘Materials and methods’. Concentrations were determined using a NanoDrop spectrophotometer. 5 μg of each sample was hybridized to 50,000 CPM of probe overnight at 42°C. Single-stranded regions were digested with RNase A/T1 and the remaining footprint: probe duplexes detected using the mirVana micro-RNA detection kit (Ambion), resolved on a 15% <t>TBE-urea</t> gel (Invitrogen), and visualized on a Storm phosphorimager (Molecular Dynamics by GE Healthcare Bio-Sciences, Pittsburgh, PA). For size markers, we end-labeled the Novex 10 bp dsDNA ladder (Invitrogen) with 32P. Over two-fold range of nuclease concentrations, the ∼30 nt peak corresponding to ribosome-protected footprints remains constant in size and intensity, indicating a lack of degradation consistent with the unchanged monosome peak height across this range of digestion conditions in ( B ). Also visible is a roughly 60 nt band which we infer to be protected by adjacent ribosomes (disomes) that sterically exclude the nuclease. This interpretation is consistent with the presence of a small disome peak in digested samples (c.f. panels B and D , and Figure 1A ). ( D ) A polysome lysate was prepared from S2 cells and resolved in 10–50% sucrose gradients, with or without prior digestion with 3 U MNase/μg total RNA ( E ) A culture of S2 cells was split into aliquots and processed using our current protocol as if they were independent samples. Total counts aligning to the coding region of each gene were tabulated in each replicate. Genes sharing at least 128 footprint counts between replicates (red) are well-correlated, demonstrating the assay is robust (see full discussion in Figure 1—figure supplement 2 ). Source data may be found in supplementary table 1 (at Dryad: Dunn et al., 2013 ). DOI: http://dx.doi.org/10.7554/eLife.01179.004
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    94
    Bio-Rad tbe urea ready gel
    Digestion with micrococcal nuclease yields a robust ribosome profiling assay. ( A ) Digestion of polysomes with RNase I degrades ribosomes. A lysate was made from S2 cells using a previous version of our protocol. Aliquots of this lysate were digested with increasing amounts of RNase I, and resolved on 10–50% sucrose gradients. As amounts of RNase I increase, the heights of all peaks—including the monosomal (80S) peak—decrease before polysomes are fully resolved to monosomes. ( B ) as in ( A ), but using micrococcal nuclease (MNase) and our current protocol. From 0.5 to 2 U MNase/μg total RNA, monosomes are resolved with no reduction in the size of the monosome peak. This result indicates that Drosophila ribosomes are stable to MNase over a broad range of concentrations, whereas the mRNA between ribosomes is digested. ( C ) Ribosome protection assay. A 320 nucleotide fragment of enolase (FlyBase accession: FBgn0000579) was amplified using oligos oJGD123 oJGD124 ( Supplementary file 2 ). A body-labeled probe against this sequence was transcribed from this template using α32P-UTP and the T7 MaxiScript kit (Ambion). S2 cell lysates were prepared as in methods and aliquoted. Aliquots were digested as in methods, except with 0, 0.5, 1, 2, 3 or 4 U MNase/μg total RNA. Monosomes were sedimented through a sucrose cushion, <t>resuspended</t> in 600 μl 10 mM Tris pH 7.0, and their RNAs extracted as in ‘Materials and methods’. Concentrations were determined using a NanoDrop spectrophotometer. 5 μg of each sample was hybridized to 50,000 CPM of probe overnight at 42°C. Single-stranded regions were digested with RNase A/T1 and the remaining footprint: probe duplexes detected using the mirVana micro-RNA detection kit (Ambion), resolved on a 15% <t>TBE-urea</t> gel (Invitrogen), and visualized on a Storm phosphorimager (Molecular Dynamics by GE Healthcare Bio-Sciences, Pittsburgh, PA). For size markers, we end-labeled the Novex 10 bp dsDNA ladder (Invitrogen) with 32P. Over two-fold range of nuclease concentrations, the ∼30 nt peak corresponding to ribosome-protected footprints remains constant in size and intensity, indicating a lack of degradation consistent with the unchanged monosome peak height across this range of digestion conditions in ( B ). Also visible is a roughly 60 nt band which we infer to be protected by adjacent ribosomes (disomes) that sterically exclude the nuclease. This interpretation is consistent with the presence of a small disome peak in digested samples (c.f. panels B and D , and Figure 1A ). ( D ) A polysome lysate was prepared from S2 cells and resolved in 10–50% sucrose gradients, with or without prior digestion with 3 U MNase/μg total RNA ( E ) A culture of S2 cells was split into aliquots and processed using our current protocol as if they were independent samples. Total counts aligning to the coding region of each gene were tabulated in each replicate. Genes sharing at least 128 footprint counts between replicates (red) are well-correlated, demonstrating the assay is robust (see full discussion in Figure 1—figure supplement 2 ). Source data may be found in supplementary table 1 (at Dryad: Dunn et al., 2013 ). DOI: http://dx.doi.org/10.7554/eLife.01179.004
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    Bio-Rad tbe gel retardation gel
    Digestion with micrococcal nuclease yields a robust ribosome profiling assay. ( A ) Digestion of polysomes with RNase I degrades ribosomes. A lysate was made from S2 cells using a previous version of our protocol. Aliquots of this lysate were digested with increasing amounts of RNase I, and resolved on 10–50% sucrose gradients. As amounts of RNase I increase, the heights of all peaks—including the monosomal (80S) peak—decrease before polysomes are fully resolved to monosomes. ( B ) as in ( A ), but using micrococcal nuclease (MNase) and our current protocol. From 0.5 to 2 U MNase/μg total RNA, monosomes are resolved with no reduction in the size of the monosome peak. This result indicates that Drosophila ribosomes are stable to MNase over a broad range of concentrations, whereas the mRNA between ribosomes is digested. ( C ) Ribosome protection assay. A 320 nucleotide fragment of enolase (FlyBase accession: FBgn0000579) was amplified using oligos oJGD123 oJGD124 ( Supplementary file 2 ). A body-labeled probe against this sequence was transcribed from this template using α32P-UTP and the T7 MaxiScript kit (Ambion). S2 cell lysates were prepared as in methods and aliquoted. Aliquots were digested as in methods, except with 0, 0.5, 1, 2, 3 or 4 U MNase/μg total RNA. Monosomes were sedimented through a sucrose cushion, <t>resuspended</t> in 600 μl 10 mM Tris pH 7.0, and their RNAs extracted as in ‘Materials and methods’. Concentrations were determined using a NanoDrop spectrophotometer. 5 μg of each sample was hybridized to 50,000 CPM of probe overnight at 42°C. Single-stranded regions were digested with RNase A/T1 and the remaining footprint: probe duplexes detected using the mirVana micro-RNA detection kit (Ambion), resolved on a 15% <t>TBE-urea</t> gel (Invitrogen), and visualized on a Storm phosphorimager (Molecular Dynamics by GE Healthcare Bio-Sciences, Pittsburgh, PA). For size markers, we end-labeled the Novex 10 bp dsDNA ladder (Invitrogen) with 32P. Over two-fold range of nuclease concentrations, the ∼30 nt peak corresponding to ribosome-protected footprints remains constant in size and intensity, indicating a lack of degradation consistent with the unchanged monosome peak height across this range of digestion conditions in ( B ). Also visible is a roughly 60 nt band which we infer to be protected by adjacent ribosomes (disomes) that sterically exclude the nuclease. This interpretation is consistent with the presence of a small disome peak in digested samples (c.f. panels B and D , and Figure 1A ). ( D ) A polysome lysate was prepared from S2 cells and resolved in 10–50% sucrose gradients, with or without prior digestion with 3 U MNase/μg total RNA ( E ) A culture of S2 cells was split into aliquots and processed using our current protocol as if they were independent samples. Total counts aligning to the coding region of each gene were tabulated in each replicate. Genes sharing at least 128 footprint counts between replicates (red) are well-correlated, demonstrating the assay is robust (see full discussion in Figure 1—figure supplement 2 ). Source data may be found in supplementary table 1 (at Dryad: Dunn et al., 2013 ). DOI: http://dx.doi.org/10.7554/eLife.01179.004
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    Thermo Fisher polyacrylaminde tbe gels
    Digestion with micrococcal nuclease yields a robust ribosome profiling assay. ( A ) Digestion of polysomes with RNase I degrades ribosomes. A lysate was made from S2 cells using a previous version of our protocol. Aliquots of this lysate were digested with increasing amounts of RNase I, and resolved on 10–50% sucrose gradients. As amounts of RNase I increase, the heights of all peaks—including the monosomal (80S) peak—decrease before polysomes are fully resolved to monosomes. ( B ) as in ( A ), but using micrococcal nuclease (MNase) and our current protocol. From 0.5 to 2 U MNase/μg total RNA, monosomes are resolved with no reduction in the size of the monosome peak. This result indicates that Drosophila ribosomes are stable to MNase over a broad range of concentrations, whereas the mRNA between ribosomes is digested. ( C ) Ribosome protection assay. A 320 nucleotide fragment of enolase (FlyBase accession: FBgn0000579) was amplified using oligos oJGD123 oJGD124 ( Supplementary file 2 ). A body-labeled probe against this sequence was transcribed from this template using α32P-UTP and the T7 MaxiScript kit (Ambion). S2 cell lysates were prepared as in methods and aliquoted. Aliquots were digested as in methods, except with 0, 0.5, 1, 2, 3 or 4 U MNase/μg total RNA. Monosomes were sedimented through a sucrose cushion, <t>resuspended</t> in 600 μl 10 mM Tris pH 7.0, and their RNAs extracted as in ‘Materials and methods’. Concentrations were determined using a NanoDrop spectrophotometer. 5 μg of each sample was hybridized to 50,000 CPM of probe overnight at 42°C. Single-stranded regions were digested with RNase A/T1 and the remaining footprint: probe duplexes detected using the mirVana micro-RNA detection kit (Ambion), resolved on a 15% <t>TBE-urea</t> gel (Invitrogen), and visualized on a Storm phosphorimager (Molecular Dynamics by GE Healthcare Bio-Sciences, Pittsburgh, PA). For size markers, we end-labeled the Novex 10 bp dsDNA ladder (Invitrogen) with 32P. Over two-fold range of nuclease concentrations, the ∼30 nt peak corresponding to ribosome-protected footprints remains constant in size and intensity, indicating a lack of degradation consistent with the unchanged monosome peak height across this range of digestion conditions in ( B ). Also visible is a roughly 60 nt band which we infer to be protected by adjacent ribosomes (disomes) that sterically exclude the nuclease. This interpretation is consistent with the presence of a small disome peak in digested samples (c.f. panels B and D , and Figure 1A ). ( D ) A polysome lysate was prepared from S2 cells and resolved in 10–50% sucrose gradients, with or without prior digestion with 3 U MNase/μg total RNA ( E ) A culture of S2 cells was split into aliquots and processed using our current protocol as if they were independent samples. Total counts aligning to the coding region of each gene were tabulated in each replicate. Genes sharing at least 128 footprint counts between replicates (red) are well-correlated, demonstrating the assay is robust (see full discussion in Figure 1—figure supplement 2 ). Source data may be found in supplementary table 1 (at Dryad: Dunn et al., 2013 ). DOI: http://dx.doi.org/10.7554/eLife.01179.004
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    Bio-Rad precasted tbe gel
    Digestion with micrococcal nuclease yields a robust ribosome profiling assay. ( A ) Digestion of polysomes with RNase I degrades ribosomes. A lysate was made from S2 cells using a previous version of our protocol. Aliquots of this lysate were digested with increasing amounts of RNase I, and resolved on 10–50% sucrose gradients. As amounts of RNase I increase, the heights of all peaks—including the monosomal (80S) peak—decrease before polysomes are fully resolved to monosomes. ( B ) as in ( A ), but using micrococcal nuclease (MNase) and our current protocol. From 0.5 to 2 U MNase/μg total RNA, monosomes are resolved with no reduction in the size of the monosome peak. This result indicates that Drosophila ribosomes are stable to MNase over a broad range of concentrations, whereas the mRNA between ribosomes is digested. ( C ) Ribosome protection assay. A 320 nucleotide fragment of enolase (FlyBase accession: FBgn0000579) was amplified using oligos oJGD123 oJGD124 ( Supplementary file 2 ). A body-labeled probe against this sequence was transcribed from this template using α32P-UTP and the T7 MaxiScript kit (Ambion). S2 cell lysates were prepared as in methods and aliquoted. Aliquots were digested as in methods, except with 0, 0.5, 1, 2, 3 or 4 U MNase/μg total RNA. Monosomes were sedimented through a sucrose cushion, <t>resuspended</t> in 600 μl 10 mM Tris pH 7.0, and their RNAs extracted as in ‘Materials and methods’. Concentrations were determined using a NanoDrop spectrophotometer. 5 μg of each sample was hybridized to 50,000 CPM of probe overnight at 42°C. Single-stranded regions were digested with RNase A/T1 and the remaining footprint: probe duplexes detected using the mirVana micro-RNA detection kit (Ambion), resolved on a 15% <t>TBE-urea</t> gel (Invitrogen), and visualized on a Storm phosphorimager (Molecular Dynamics by GE Healthcare Bio-Sciences, Pittsburgh, PA). For size markers, we end-labeled the Novex 10 bp dsDNA ladder (Invitrogen) with 32P. Over two-fold range of nuclease concentrations, the ∼30 nt peak corresponding to ribosome-protected footprints remains constant in size and intensity, indicating a lack of degradation consistent with the unchanged monosome peak height across this range of digestion conditions in ( B ). Also visible is a roughly 60 nt band which we infer to be protected by adjacent ribosomes (disomes) that sterically exclude the nuclease. This interpretation is consistent with the presence of a small disome peak in digested samples (c.f. panels B and D , and Figure 1A ). ( D ) A polysome lysate was prepared from S2 cells and resolved in 10–50% sucrose gradients, with or without prior digestion with 3 U MNase/μg total RNA ( E ) A culture of S2 cells was split into aliquots and processed using our current protocol as if they were independent samples. Total counts aligning to the coding region of each gene were tabulated in each replicate. Genes sharing at least 128 footprint counts between replicates (red) are well-correlated, demonstrating the assay is robust (see full discussion in Figure 1—figure supplement 2 ). Source data may be found in supplementary table 1 (at Dryad: Dunn et al., 2013 ). DOI: http://dx.doi.org/10.7554/eLife.01179.004
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    Bio-Rad tbe precast gel
    Digestion with micrococcal nuclease yields a robust ribosome profiling assay. ( A ) Digestion of polysomes with RNase I degrades ribosomes. A lysate was made from S2 cells using a previous version of our protocol. Aliquots of this lysate were digested with increasing amounts of RNase I, and resolved on 10–50% sucrose gradients. As amounts of RNase I increase, the heights of all peaks—including the monosomal (80S) peak—decrease before polysomes are fully resolved to monosomes. ( B ) as in ( A ), but using micrococcal nuclease (MNase) and our current protocol. From 0.5 to 2 U MNase/μg total RNA, monosomes are resolved with no reduction in the size of the monosome peak. This result indicates that Drosophila ribosomes are stable to MNase over a broad range of concentrations, whereas the mRNA between ribosomes is digested. ( C ) Ribosome protection assay. A 320 nucleotide fragment of enolase (FlyBase accession: FBgn0000579) was amplified using oligos oJGD123 oJGD124 ( Supplementary file 2 ). A body-labeled probe against this sequence was transcribed from this template using α32P-UTP and the T7 MaxiScript kit (Ambion). S2 cell lysates were prepared as in methods and aliquoted. Aliquots were digested as in methods, except with 0, 0.5, 1, 2, 3 or 4 U MNase/μg total RNA. Monosomes were sedimented through a sucrose cushion, <t>resuspended</t> in 600 μl 10 mM Tris pH 7.0, and their RNAs extracted as in ‘Materials and methods’. Concentrations were determined using a NanoDrop spectrophotometer. 5 μg of each sample was hybridized to 50,000 CPM of probe overnight at 42°C. Single-stranded regions were digested with RNase A/T1 and the remaining footprint: probe duplexes detected using the mirVana micro-RNA detection kit (Ambion), resolved on a 15% <t>TBE-urea</t> gel (Invitrogen), and visualized on a Storm phosphorimager (Molecular Dynamics by GE Healthcare Bio-Sciences, Pittsburgh, PA). For size markers, we end-labeled the Novex 10 bp dsDNA ladder (Invitrogen) with 32P. Over two-fold range of nuclease concentrations, the ∼30 nt peak corresponding to ribosome-protected footprints remains constant in size and intensity, indicating a lack of degradation consistent with the unchanged monosome peak height across this range of digestion conditions in ( B ). Also visible is a roughly 60 nt band which we infer to be protected by adjacent ribosomes (disomes) that sterically exclude the nuclease. This interpretation is consistent with the presence of a small disome peak in digested samples (c.f. panels B and D , and Figure 1A ). ( D ) A polysome lysate was prepared from S2 cells and resolved in 10–50% sucrose gradients, with or without prior digestion with 3 U MNase/μg total RNA ( E ) A culture of S2 cells was split into aliquots and processed using our current protocol as if they were independent samples. Total counts aligning to the coding region of each gene were tabulated in each replicate. Genes sharing at least 128 footprint counts between replicates (red) are well-correlated, demonstrating the assay is robust (see full discussion in Figure 1—figure supplement 2 ). Source data may be found in supplementary table 1 (at Dryad: Dunn et al., 2013 ). DOI: http://dx.doi.org/10.7554/eLife.01179.004
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    Bio-Rad tbe readymade gels
    Digestion with micrococcal nuclease yields a robust ribosome profiling assay. ( A ) Digestion of polysomes with RNase I degrades ribosomes. A lysate was made from S2 cells using a previous version of our protocol. Aliquots of this lysate were digested with increasing amounts of RNase I, and resolved on 10–50% sucrose gradients. As amounts of RNase I increase, the heights of all peaks—including the monosomal (80S) peak—decrease before polysomes are fully resolved to monosomes. ( B ) as in ( A ), but using micrococcal nuclease (MNase) and our current protocol. From 0.5 to 2 U MNase/μg total RNA, monosomes are resolved with no reduction in the size of the monosome peak. This result indicates that Drosophila ribosomes are stable to MNase over a broad range of concentrations, whereas the mRNA between ribosomes is digested. ( C ) Ribosome protection assay. A 320 nucleotide fragment of enolase (FlyBase accession: FBgn0000579) was amplified using oligos oJGD123 oJGD124 ( Supplementary file 2 ). A body-labeled probe against this sequence was transcribed from this template using α32P-UTP and the T7 MaxiScript kit (Ambion). S2 cell lysates were prepared as in methods and aliquoted. Aliquots were digested as in methods, except with 0, 0.5, 1, 2, 3 or 4 U MNase/μg total RNA. Monosomes were sedimented through a sucrose cushion, <t>resuspended</t> in 600 μl 10 mM Tris pH 7.0, and their RNAs extracted as in ‘Materials and methods’. Concentrations were determined using a NanoDrop spectrophotometer. 5 μg of each sample was hybridized to 50,000 CPM of probe overnight at 42°C. Single-stranded regions were digested with RNase A/T1 and the remaining footprint: probe duplexes detected using the mirVana micro-RNA detection kit (Ambion), resolved on a 15% <t>TBE-urea</t> gel (Invitrogen), and visualized on a Storm phosphorimager (Molecular Dynamics by GE Healthcare Bio-Sciences, Pittsburgh, PA). For size markers, we end-labeled the Novex 10 bp dsDNA ladder (Invitrogen) with 32P. Over two-fold range of nuclease concentrations, the ∼30 nt peak corresponding to ribosome-protected footprints remains constant in size and intensity, indicating a lack of degradation consistent with the unchanged monosome peak height across this range of digestion conditions in ( B ). Also visible is a roughly 60 nt band which we infer to be protected by adjacent ribosomes (disomes) that sterically exclude the nuclease. This interpretation is consistent with the presence of a small disome peak in digested samples (c.f. panels B and D , and Figure 1A ). ( D ) A polysome lysate was prepared from S2 cells and resolved in 10–50% sucrose gradients, with or without prior digestion with 3 U MNase/μg total RNA ( E ) A culture of S2 cells was split into aliquots and processed using our current protocol as if they were independent samples. Total counts aligning to the coding region of each gene were tabulated in each replicate. Genes sharing at least 128 footprint counts between replicates (red) are well-correlated, demonstrating the assay is robust (see full discussion in Figure 1—figure supplement 2 ). Source data may be found in supplementary table 1 (at Dryad: Dunn et al., 2013 ). DOI: http://dx.doi.org/10.7554/eLife.01179.004
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    Bio-Rad mini protean tbe precast gel
    Digestion with micrococcal nuclease yields a robust ribosome profiling assay. ( A ) Digestion of polysomes with RNase I degrades ribosomes. A lysate was made from S2 cells using a previous version of our protocol. Aliquots of this lysate were digested with increasing amounts of RNase I, and resolved on 10–50% sucrose gradients. As amounts of RNase I increase, the heights of all peaks—including the monosomal (80S) peak—decrease before polysomes are fully resolved to monosomes. ( B ) as in ( A ), but using micrococcal nuclease (MNase) and our current protocol. From 0.5 to 2 U MNase/μg total RNA, monosomes are resolved with no reduction in the size of the monosome peak. This result indicates that Drosophila ribosomes are stable to MNase over a broad range of concentrations, whereas the mRNA between ribosomes is digested. ( C ) Ribosome protection assay. A 320 nucleotide fragment of enolase (FlyBase accession: FBgn0000579) was amplified using oligos oJGD123 oJGD124 ( Supplementary file 2 ). A body-labeled probe against this sequence was transcribed from this template using α32P-UTP and the T7 MaxiScript kit (Ambion). S2 cell lysates were prepared as in methods and aliquoted. Aliquots were digested as in methods, except with 0, 0.5, 1, 2, 3 or 4 U MNase/μg total RNA. Monosomes were sedimented through a sucrose cushion, <t>resuspended</t> in 600 μl 10 mM Tris pH 7.0, and their RNAs extracted as in ‘Materials and methods’. Concentrations were determined using a NanoDrop spectrophotometer. 5 μg of each sample was hybridized to 50,000 CPM of probe overnight at 42°C. Single-stranded regions were digested with RNase A/T1 and the remaining footprint: probe duplexes detected using the mirVana micro-RNA detection kit (Ambion), resolved on a 15% <t>TBE-urea</t> gel (Invitrogen), and visualized on a Storm phosphorimager (Molecular Dynamics by GE Healthcare Bio-Sciences, Pittsburgh, PA). For size markers, we end-labeled the Novex 10 bp dsDNA ladder (Invitrogen) with 32P. Over two-fold range of nuclease concentrations, the ∼30 nt peak corresponding to ribosome-protected footprints remains constant in size and intensity, indicating a lack of degradation consistent with the unchanged monosome peak height across this range of digestion conditions in ( B ). Also visible is a roughly 60 nt band which we infer to be protected by adjacent ribosomes (disomes) that sterically exclude the nuclease. This interpretation is consistent with the presence of a small disome peak in digested samples (c.f. panels B and D , and Figure 1A ). ( D ) A polysome lysate was prepared from S2 cells and resolved in 10–50% sucrose gradients, with or without prior digestion with 3 U MNase/μg total RNA ( E ) A culture of S2 cells was split into aliquots and processed using our current protocol as if they were independent samples. Total counts aligning to the coding region of each gene were tabulated in each replicate. Genes sharing at least 128 footprint counts between replicates (red) are well-correlated, demonstrating the assay is robust (see full discussion in Figure 1—figure supplement 2 ). Source data may be found in supplementary table 1 (at Dryad: Dunn et al., 2013 ). DOI: http://dx.doi.org/10.7554/eLife.01179.004
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    Thermo Fisher acrylamide tbe gel
    Digestion with micrococcal nuclease yields a robust ribosome profiling assay. ( A ) Digestion of polysomes with RNase I degrades ribosomes. A lysate was made from S2 cells using a previous version of our protocol. Aliquots of this lysate were digested with increasing amounts of RNase I, and resolved on 10–50% sucrose gradients. As amounts of RNase I increase, the heights of all peaks—including the monosomal (80S) peak—decrease before polysomes are fully resolved to monosomes. ( B ) as in ( A ), but using micrococcal nuclease (MNase) and our current protocol. From 0.5 to 2 U MNase/μg total RNA, monosomes are resolved with no reduction in the size of the monosome peak. This result indicates that Drosophila ribosomes are stable to MNase over a broad range of concentrations, whereas the mRNA between ribosomes is digested. ( C ) Ribosome protection assay. A 320 nucleotide fragment of enolase (FlyBase accession: FBgn0000579) was amplified using oligos oJGD123 oJGD124 ( Supplementary file 2 ). A body-labeled probe against this sequence was transcribed from this template using α32P-UTP and the T7 MaxiScript kit (Ambion). S2 cell lysates were prepared as in methods and aliquoted. Aliquots were digested as in methods, except with 0, 0.5, 1, 2, 3 or 4 U MNase/μg total RNA. Monosomes were sedimented through a sucrose cushion, <t>resuspended</t> in 600 μl 10 mM Tris pH 7.0, and their RNAs extracted as in ‘Materials and methods’. Concentrations were determined using a NanoDrop spectrophotometer. 5 μg of each sample was hybridized to 50,000 CPM of probe overnight at 42°C. Single-stranded regions were digested with RNase A/T1 and the remaining footprint: probe duplexes detected using the mirVana micro-RNA detection kit (Ambion), resolved on a 15% <t>TBE-urea</t> gel (Invitrogen), and visualized on a Storm phosphorimager (Molecular Dynamics by GE Healthcare Bio-Sciences, Pittsburgh, PA). For size markers, we end-labeled the Novex 10 bp dsDNA ladder (Invitrogen) with 32P. Over two-fold range of nuclease concentrations, the ∼30 nt peak corresponding to ribosome-protected footprints remains constant in size and intensity, indicating a lack of degradation consistent with the unchanged monosome peak height across this range of digestion conditions in ( B ). Also visible is a roughly 60 nt band which we infer to be protected by adjacent ribosomes (disomes) that sterically exclude the nuclease. This interpretation is consistent with the presence of a small disome peak in digested samples (c.f. panels B and D , and Figure 1A ). ( D ) A polysome lysate was prepared from S2 cells and resolved in 10–50% sucrose gradients, with or without prior digestion with 3 U MNase/μg total RNA ( E ) A culture of S2 cells was split into aliquots and processed using our current protocol as if they were independent samples. Total counts aligning to the coding region of each gene were tabulated in each replicate. Genes sharing at least 128 footprint counts between replicates (red) are well-correlated, demonstrating the assay is robust (see full discussion in Figure 1—figure supplement 2 ). Source data may be found in supplementary table 1 (at Dryad: Dunn et al., 2013 ). DOI: http://dx.doi.org/10.7554/eLife.01179.004
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    Digestion with micrococcal nuclease yields a robust ribosome profiling assay. ( A ) Digestion of polysomes with RNase I degrades ribosomes. A lysate was made from S2 cells using a previous version of our protocol. Aliquots of this lysate were digested with increasing amounts of RNase I, and resolved on 10–50% sucrose gradients. As amounts of RNase I increase, the heights of all peaks—including the monosomal (80S) peak—decrease before polysomes are fully resolved to monosomes. ( B ) as in ( A ), but using micrococcal nuclease (MNase) and our current protocol. From 0.5 to 2 U MNase/μg total RNA, monosomes are resolved with no reduction in the size of the monosome peak. This result indicates that Drosophila ribosomes are stable to MNase over a broad range of concentrations, whereas the mRNA between ribosomes is digested. ( C ) Ribosome protection assay. A 320 nucleotide fragment of enolase (FlyBase accession: FBgn0000579) was amplified using oligos oJGD123 oJGD124 ( Supplementary file 2 ). A body-labeled probe against this sequence was transcribed from this template using α32P-UTP and the T7 MaxiScript kit (Ambion). S2 cell lysates were prepared as in methods and aliquoted. Aliquots were digested as in methods, except with 0, 0.5, 1, 2, 3 or 4 U MNase/μg total RNA. Monosomes were sedimented through a sucrose cushion, resuspended in 600 μl 10 mM Tris pH 7.0, and their RNAs extracted as in ‘Materials and methods’. Concentrations were determined using a NanoDrop spectrophotometer. 5 μg of each sample was hybridized to 50,000 CPM of probe overnight at 42°C. Single-stranded regions were digested with RNase A/T1 and the remaining footprint: probe duplexes detected using the mirVana micro-RNA detection kit (Ambion), resolved on a 15% TBE-urea gel (Invitrogen), and visualized on a Storm phosphorimager (Molecular Dynamics by GE Healthcare Bio-Sciences, Pittsburgh, PA). For size markers, we end-labeled the Novex 10 bp dsDNA ladder (Invitrogen) with 32P. Over two-fold range of nuclease concentrations, the ∼30 nt peak corresponding to ribosome-protected footprints remains constant in size and intensity, indicating a lack of degradation consistent with the unchanged monosome peak height across this range of digestion conditions in ( B ). Also visible is a roughly 60 nt band which we infer to be protected by adjacent ribosomes (disomes) that sterically exclude the nuclease. This interpretation is consistent with the presence of a small disome peak in digested samples (c.f. panels B and D , and Figure 1A ). ( D ) A polysome lysate was prepared from S2 cells and resolved in 10–50% sucrose gradients, with or without prior digestion with 3 U MNase/μg total RNA ( E ) A culture of S2 cells was split into aliquots and processed using our current protocol as if they were independent samples. Total counts aligning to the coding region of each gene were tabulated in each replicate. Genes sharing at least 128 footprint counts between replicates (red) are well-correlated, demonstrating the assay is robust (see full discussion in Figure 1—figure supplement 2 ). Source data may be found in supplementary table 1 (at Dryad: Dunn et al., 2013 ). DOI: http://dx.doi.org/10.7554/eLife.01179.004

    Journal: eLife

    Article Title: Ribosome profiling reveals pervasive and regulated stop codon readthrough in Drosophila melanogaster

    doi: 10.7554/eLife.01179

    Figure Lengend Snippet: Digestion with micrococcal nuclease yields a robust ribosome profiling assay. ( A ) Digestion of polysomes with RNase I degrades ribosomes. A lysate was made from S2 cells using a previous version of our protocol. Aliquots of this lysate were digested with increasing amounts of RNase I, and resolved on 10–50% sucrose gradients. As amounts of RNase I increase, the heights of all peaks—including the monosomal (80S) peak—decrease before polysomes are fully resolved to monosomes. ( B ) as in ( A ), but using micrococcal nuclease (MNase) and our current protocol. From 0.5 to 2 U MNase/μg total RNA, monosomes are resolved with no reduction in the size of the monosome peak. This result indicates that Drosophila ribosomes are stable to MNase over a broad range of concentrations, whereas the mRNA between ribosomes is digested. ( C ) Ribosome protection assay. A 320 nucleotide fragment of enolase (FlyBase accession: FBgn0000579) was amplified using oligos oJGD123 oJGD124 ( Supplementary file 2 ). A body-labeled probe against this sequence was transcribed from this template using α32P-UTP and the T7 MaxiScript kit (Ambion). S2 cell lysates were prepared as in methods and aliquoted. Aliquots were digested as in methods, except with 0, 0.5, 1, 2, 3 or 4 U MNase/μg total RNA. Monosomes were sedimented through a sucrose cushion, resuspended in 600 μl 10 mM Tris pH 7.0, and their RNAs extracted as in ‘Materials and methods’. Concentrations were determined using a NanoDrop spectrophotometer. 5 μg of each sample was hybridized to 50,000 CPM of probe overnight at 42°C. Single-stranded regions were digested with RNase A/T1 and the remaining footprint: probe duplexes detected using the mirVana micro-RNA detection kit (Ambion), resolved on a 15% TBE-urea gel (Invitrogen), and visualized on a Storm phosphorimager (Molecular Dynamics by GE Healthcare Bio-Sciences, Pittsburgh, PA). For size markers, we end-labeled the Novex 10 bp dsDNA ladder (Invitrogen) with 32P. Over two-fold range of nuclease concentrations, the ∼30 nt peak corresponding to ribosome-protected footprints remains constant in size and intensity, indicating a lack of degradation consistent with the unchanged monosome peak height across this range of digestion conditions in ( B ). Also visible is a roughly 60 nt band which we infer to be protected by adjacent ribosomes (disomes) that sterically exclude the nuclease. This interpretation is consistent with the presence of a small disome peak in digested samples (c.f. panels B and D , and Figure 1A ). ( D ) A polysome lysate was prepared from S2 cells and resolved in 10–50% sucrose gradients, with or without prior digestion with 3 U MNase/μg total RNA ( E ) A culture of S2 cells was split into aliquots and processed using our current protocol as if they were independent samples. Total counts aligning to the coding region of each gene were tabulated in each replicate. Genes sharing at least 128 footprint counts between replicates (red) are well-correlated, demonstrating the assay is robust (see full discussion in Figure 1—figure supplement 2 ). Source data may be found in supplementary table 1 (at Dryad: Dunn et al., 2013 ). DOI: http://dx.doi.org/10.7554/eLife.01179.004

    Article Snippet: Amplification products were size-selected on 8% TBE gels (Invitrogen), eluted, precipitated, and resuspended in 10 µl 10 mM Tris, pH 8.0.

    Techniques: Amplification, Labeling, Sequencing, Spectrophotometry, RNA Detection

    DNA binding properties of wild-type and mutants PB Cysteine-Rich Domain. ( A ) Wild type PB CRD (GST-PB(537–594)) formed different types of complexes in the presence of the LE1–35 and Random substrate. Complexes were assembled in the presence of LE1–35-Cy3 or Random-Cy3 substrates (100 nM) and 0.33 μM, 1 or 3 μM concentration of GST-PB(537–594) before analysis on a 5% acrylamide, 0.5× TBE gel. ( B ) Wild-type or mutants GST-PB(537–594)/LE1–35 complexes were assembled in the presence of 100 nM of LE1–35-Cy3 substrate and 0.03 μM, 0.11, 0.33, 1 or 3 μM of GST fused PB(537–594). Complexes were further analysed by EMSA on a 5% acrylamide, 0.5× TBE gel.

    Journal: Nucleic Acids Research

    Article Title: Sequence-specific DNA binding activity of the cross-brace zinc finger motif of the piggyBac transposase

    doi: 10.1093/nar/gky044

    Figure Lengend Snippet: DNA binding properties of wild-type and mutants PB Cysteine-Rich Domain. ( A ) Wild type PB CRD (GST-PB(537–594)) formed different types of complexes in the presence of the LE1–35 and Random substrate. Complexes were assembled in the presence of LE1–35-Cy3 or Random-Cy3 substrates (100 nM) and 0.33 μM, 1 or 3 μM concentration of GST-PB(537–594) before analysis on a 5% acrylamide, 0.5× TBE gel. ( B ) Wild-type or mutants GST-PB(537–594)/LE1–35 complexes were assembled in the presence of 100 nM of LE1–35-Cy3 substrate and 0.03 μM, 0.11, 0.33, 1 or 3 μM of GST fused PB(537–594). Complexes were further analysed by EMSA on a 5% acrylamide, 0.5× TBE gel.

    Article Snippet: Samples were loaded onto a 10% acrylamide 1× TBE gel (Invitrogen), and run at 100 V at 4°C for 2 h. Gels were visualized using a GE Typhoon Trio variable mode imager.

    Techniques: Binding Assay, Concentration Assay