sigma superase in  (Thermo Fisher)


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    Structured Review

    Thermo Fisher sigma superase in
    Sigma Superase In, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sigma superase in/product/Thermo Fisher
    Average 92 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    sigma superase in - by Bioz Stars, 2020-09
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    Related Articles

    Polymerase Chain Reaction:

    Article Title: A longitudinal systems immunologic investigation of acute Zika virus infection in an individual infected while traveling to Caracas, Venezuela
    Article Snippet: .. 10 μL purified RNA was mixed with 5.5 μL of SMARTScribe 5X First-Strand Buffer (Clontech), 1 μL polyT-RT primer (2.5 μM, 5’-AAGCAGTGGTATCAACGCAGAGTAC(T30)VN, 0.5 μL SUPERase-IN (Ambion), 4 μL dNTP mix (10 mM, Invitrogen), 0.5 μL DTT (20 mM, Clontech) and 2 μL Betaine solution (5 M, Sigma), incubated 50°C 3 min. 3.9 μL of first strand mix, containing 0.2 μL 1% Tween-20, 0.32 μL MgCl2 (500 mM), 0.88 μL Betaine solution (5 M, Sigma), 0.5 μL (5 M, Sigma) SUPERase-IN (Ambion) and 2 μL SMARTScribe Reverse Transcriptase (100 U/μL Clontech) was added and incubated one cycle 25°C 3 min., 42°C 60 min. 1.62 μL template switch (TS) reaction mix containing 0.8 μL biotin-TS oligo (10 μM, Biotin-5’-AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3’), 0.5 μL SMARTScribe Reverse Transcriptase (100 U/μL Clontech) and 0.32 μL SMARTScribe 5X First-Strand Buffer (Clontech) was added, then incubated at 50°C 2 min., 42°C 80 min., 70°C 10 min. 14.8 μL second strand synthesis, pre-amplification mix containing 1 μL pre-amp oligo (10 μM, 5’AAGCAGTGGTATCAACGCAGAGT-3’), 8.8 μL KAPA HiFi Fidelity Buffer (5X, KAPA Biosystems), 3.5 μL dNTP mix (10 mM, Invitrogen) and 1.5 μL KAPA HiFi HotStart DNA Polymerase (1U/μL, KAPA Biosystems), was added, then amplified by PCR: 95°C 3 min., 5 cycles 98°C 20 sec, 67°C 15 sec and 72°C 6 min, final extension 72°C 5 min. .. The synthesized dsDNA was purified using Sera-Mag Speedbeads (Thermo Fisher Scientific) with final 8.4% PEG8000, 1.1M NaCl, then eluted with 13 μL UltraPure water (Invitrogen).

    Incubation:

    Article Title: A longitudinal systems immunologic investigation of acute Zika virus infection in an individual infected while traveling to Caracas, Venezuela
    Article Snippet: .. 10 μL purified RNA was mixed with 5.5 μL of SMARTScribe 5X First-Strand Buffer (Clontech), 1 μL polyT-RT primer (2.5 μM, 5’-AAGCAGTGGTATCAACGCAGAGTAC(T30)VN, 0.5 μL SUPERase-IN (Ambion), 4 μL dNTP mix (10 mM, Invitrogen), 0.5 μL DTT (20 mM, Clontech) and 2 μL Betaine solution (5 M, Sigma), incubated 50°C 3 min. 3.9 μL of first strand mix, containing 0.2 μL 1% Tween-20, 0.32 μL MgCl2 (500 mM), 0.88 μL Betaine solution (5 M, Sigma), 0.5 μL (5 M, Sigma) SUPERase-IN (Ambion) and 2 μL SMARTScribe Reverse Transcriptase (100 U/μL Clontech) was added and incubated one cycle 25°C 3 min., 42°C 60 min. 1.62 μL template switch (TS) reaction mix containing 0.8 μL biotin-TS oligo (10 μM, Biotin-5’-AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3’), 0.5 μL SMARTScribe Reverse Transcriptase (100 U/μL Clontech) and 0.32 μL SMARTScribe 5X First-Strand Buffer (Clontech) was added, then incubated at 50°C 2 min., 42°C 80 min., 70°C 10 min. 14.8 μL second strand synthesis, pre-amplification mix containing 1 μL pre-amp oligo (10 μM, 5’AAGCAGTGGTATCAACGCAGAGT-3’), 8.8 μL KAPA HiFi Fidelity Buffer (5X, KAPA Biosystems), 3.5 μL dNTP mix (10 mM, Invitrogen) and 1.5 μL KAPA HiFi HotStart DNA Polymerase (1U/μL, KAPA Biosystems), was added, then amplified by PCR: 95°C 3 min., 5 cycles 98°C 20 sec, 67°C 15 sec and 72°C 6 min, final extension 72°C 5 min. .. The synthesized dsDNA was purified using Sera-Mag Speedbeads (Thermo Fisher Scientific) with final 8.4% PEG8000, 1.1M NaCl, then eluted with 13 μL UltraPure water (Invitrogen).

    Amplification:

    Article Title: A longitudinal systems immunologic investigation of acute Zika virus infection in an individual infected while traveling to Caracas, Venezuela
    Article Snippet: .. 10 μL purified RNA was mixed with 5.5 μL of SMARTScribe 5X First-Strand Buffer (Clontech), 1 μL polyT-RT primer (2.5 μM, 5’-AAGCAGTGGTATCAACGCAGAGTAC(T30)VN, 0.5 μL SUPERase-IN (Ambion), 4 μL dNTP mix (10 mM, Invitrogen), 0.5 μL DTT (20 mM, Clontech) and 2 μL Betaine solution (5 M, Sigma), incubated 50°C 3 min. 3.9 μL of first strand mix, containing 0.2 μL 1% Tween-20, 0.32 μL MgCl2 (500 mM), 0.88 μL Betaine solution (5 M, Sigma), 0.5 μL (5 M, Sigma) SUPERase-IN (Ambion) and 2 μL SMARTScribe Reverse Transcriptase (100 U/μL Clontech) was added and incubated one cycle 25°C 3 min., 42°C 60 min. 1.62 μL template switch (TS) reaction mix containing 0.8 μL biotin-TS oligo (10 μM, Biotin-5’-AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3’), 0.5 μL SMARTScribe Reverse Transcriptase (100 U/μL Clontech) and 0.32 μL SMARTScribe 5X First-Strand Buffer (Clontech) was added, then incubated at 50°C 2 min., 42°C 80 min., 70°C 10 min. 14.8 μL second strand synthesis, pre-amplification mix containing 1 μL pre-amp oligo (10 μM, 5’AAGCAGTGGTATCAACGCAGAGT-3’), 8.8 μL KAPA HiFi Fidelity Buffer (5X, KAPA Biosystems), 3.5 μL dNTP mix (10 mM, Invitrogen) and 1.5 μL KAPA HiFi HotStart DNA Polymerase (1U/μL, KAPA Biosystems), was added, then amplified by PCR: 95°C 3 min., 5 cycles 98°C 20 sec, 67°C 15 sec and 72°C 6 min, final extension 72°C 5 min. .. The synthesized dsDNA was purified using Sera-Mag Speedbeads (Thermo Fisher Scientific) with final 8.4% PEG8000, 1.1M NaCl, then eluted with 13 μL UltraPure water (Invitrogen).

    Size-exclusion Chromatography:

    Article Title: A longitudinal systems immunologic investigation of acute Zika virus infection in an individual infected while traveling to Caracas, Venezuela
    Article Snippet: .. 10 μL purified RNA was mixed with 5.5 μL of SMARTScribe 5X First-Strand Buffer (Clontech), 1 μL polyT-RT primer (2.5 μM, 5’-AAGCAGTGGTATCAACGCAGAGTAC(T30)VN, 0.5 μL SUPERase-IN (Ambion), 4 μL dNTP mix (10 mM, Invitrogen), 0.5 μL DTT (20 mM, Clontech) and 2 μL Betaine solution (5 M, Sigma), incubated 50°C 3 min. 3.9 μL of first strand mix, containing 0.2 μL 1% Tween-20, 0.32 μL MgCl2 (500 mM), 0.88 μL Betaine solution (5 M, Sigma), 0.5 μL (5 M, Sigma) SUPERase-IN (Ambion) and 2 μL SMARTScribe Reverse Transcriptase (100 U/μL Clontech) was added and incubated one cycle 25°C 3 min., 42°C 60 min. 1.62 μL template switch (TS) reaction mix containing 0.8 μL biotin-TS oligo (10 μM, Biotin-5’-AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3’), 0.5 μL SMARTScribe Reverse Transcriptase (100 U/μL Clontech) and 0.32 μL SMARTScribe 5X First-Strand Buffer (Clontech) was added, then incubated at 50°C 2 min., 42°C 80 min., 70°C 10 min. 14.8 μL second strand synthesis, pre-amplification mix containing 1 μL pre-amp oligo (10 μM, 5’AAGCAGTGGTATCAACGCAGAGT-3’), 8.8 μL KAPA HiFi Fidelity Buffer (5X, KAPA Biosystems), 3.5 μL dNTP mix (10 mM, Invitrogen) and 1.5 μL KAPA HiFi HotStart DNA Polymerase (1U/μL, KAPA Biosystems), was added, then amplified by PCR: 95°C 3 min., 5 cycles 98°C 20 sec, 67°C 15 sec and 72°C 6 min, final extension 72°C 5 min. .. The synthesized dsDNA was purified using Sera-Mag Speedbeads (Thermo Fisher Scientific) with final 8.4% PEG8000, 1.1M NaCl, then eluted with 13 μL UltraPure water (Invitrogen).

    Purification:

    Article Title: A longitudinal systems immunologic investigation of acute Zika virus infection in an individual infected while traveling to Caracas, Venezuela
    Article Snippet: .. 10 μL purified RNA was mixed with 5.5 μL of SMARTScribe 5X First-Strand Buffer (Clontech), 1 μL polyT-RT primer (2.5 μM, 5’-AAGCAGTGGTATCAACGCAGAGTAC(T30)VN, 0.5 μL SUPERase-IN (Ambion), 4 μL dNTP mix (10 mM, Invitrogen), 0.5 μL DTT (20 mM, Clontech) and 2 μL Betaine solution (5 M, Sigma), incubated 50°C 3 min. 3.9 μL of first strand mix, containing 0.2 μL 1% Tween-20, 0.32 μL MgCl2 (500 mM), 0.88 μL Betaine solution (5 M, Sigma), 0.5 μL (5 M, Sigma) SUPERase-IN (Ambion) and 2 μL SMARTScribe Reverse Transcriptase (100 U/μL Clontech) was added and incubated one cycle 25°C 3 min., 42°C 60 min. 1.62 μL template switch (TS) reaction mix containing 0.8 μL biotin-TS oligo (10 μM, Biotin-5’-AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3’), 0.5 μL SMARTScribe Reverse Transcriptase (100 U/μL Clontech) and 0.32 μL SMARTScribe 5X First-Strand Buffer (Clontech) was added, then incubated at 50°C 2 min., 42°C 80 min., 70°C 10 min. 14.8 μL second strand synthesis, pre-amplification mix containing 1 μL pre-amp oligo (10 μM, 5’AAGCAGTGGTATCAACGCAGAGT-3’), 8.8 μL KAPA HiFi Fidelity Buffer (5X, KAPA Biosystems), 3.5 μL dNTP mix (10 mM, Invitrogen) and 1.5 μL KAPA HiFi HotStart DNA Polymerase (1U/μL, KAPA Biosystems), was added, then amplified by PCR: 95°C 3 min., 5 cycles 98°C 20 sec, 67°C 15 sec and 72°C 6 min, final extension 72°C 5 min. .. The synthesized dsDNA was purified using Sera-Mag Speedbeads (Thermo Fisher Scientific) with final 8.4% PEG8000, 1.1M NaCl, then eluted with 13 μL UltraPure water (Invitrogen).

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    Thermo Fisher rnase t1
    RPF GC content is <t>RNase-species</t> independent. ( A ) 3.8 A 260 homogenate from hippocampi of one P35 male mouse was digested with 100ng RNase A (Sigma, # R4875) + 60U <t>RNase</t> T1 (Thermo Fisher Scientific, #EN0542)/ A 260 , at 25°C for 30min and applied to a 10–50% (w/v) sucrose gradient. ( B ) 3.8 A 260 homogenate from hippocampi of one P35 mouse was digested with 5U RNase I (Ambion, #AM2294)/ A 260 , at 25°C for 45min and applied to a 10–50% (w/v) sucrose gradient. ( C ) Nucleotide composition at each position of RPFs mapped to CDS from ribosomes in (A). ( D ) Nucleotide composition at each position of RPFs mapped to CDS from ribosomes in (B). ( E ) Nucleotide composition at each position of RPFs mapped to CDS from mouse embryonic stem cells (mESCs) (data from Ingolia et al. ) ( 16 ). A 600 μl aliquot of lysate was treated with 15 μl RNase I at 100 U/μl for 45 min at 25°C. ( F ) Nucleotide composition at each position of RPFs mapped to CDS from human embryonic stem cell (hESC)-derived neurons (data from Grabole et al. ) ( 42 ). 5 U TruSeq Ribo Profile Nuclease/ A 260 at 25°C for 45 min.
    Rnase T1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 518 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase t1/product/Thermo Fisher
    Average 99 stars, based on 518 article reviews
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    99
    Thermo Fisher emsa buffer
    Optimization of fluorescence labeling of MAGIC Factor and MAGIC Probes. ( A ) Labeling strategy of MAGIC Factor. MAGIC Factor was fluorescently labeled with increasing amounts of Alexa Fluor 488 SDP Ester and run on a 12% SDS-PAGE gel after purification by size-exclusion chromatography. The ratios refer to dye:protein molar ratios during fluorescent labeling. The control reaction was performed with DMSO instead of the dye. The same gel was imaged for native Alexa Fluor 488 fluorescence (top gel) and after staining all protein with Sypro Orange (bottom gel). ( B ) Electrophoretic Mobility Shift Assay <t>(EMSA)</t> of fluorescently labeled MAGIC Factor. Proteins from A were reacted with Alexa Fluor 647-labeled dsRNA and run on a native gel to visualize the binding affinities of the proteins. The gel was visualized in the <t>RNA</t> channel, protein channel and FRET channel. Note that the appearance of MAGIC Factor as multiple bands is likely due to the use of an NHS-ester dye to attach Alexa Fluor 488 to the protein. Dependent on the exact location of the fluorophore molecule, each single protein molecule likely runs differently on the native polyacrylamide gel. ( C ) Quantitative assessment of the EMSA with respect to the relative shift of the dsRNA, the corrected FRET (cFRET) intensity of shifted dsRNA and the cFRET/shift ratio. ( D ) Unlabeled and fluorescently labeled MAGIC Factor were affinity purified using dsRNA-coupled agarose beads. The proteins were reacted with the beads for 1 hr, washed three times with binding buffer and then gradually eluted with increasing concentrations of KCl. At the end, remaining proteins were eluted with 1x SDS-PAGE sample buffer and all samples run on a 12% SDS-PAGE gel. As a control experiment, fluorescently labeled MAGIC Factor was reacted with agarose beads in the absence of dsRNA. Gels were imaged for native Alexa Fluor 488 fluorescence (right gel) and after staining all protein (left gel). ( E ) Effect of degree of RNA fluorescent labeling on probe hybridization kinetics. Fluorescently labeled RNA probes were gel purified to obtain one to four labeled RNA probes. They were then reacted with unlabeled sense probes to generate dsRNA in TEN 100 buffer (tris, EDTA, sodium chloride) at 95°C (positive control) or physiologic buffer resembling cytoplasmic ionic concentrations at 37°C for 30 min or 2 hr. ssRNA are shown as controls. Representative native 20% polyacrylamide gels are shown. ( F ) Quantification of RNA fluorescence intensities from ( E ). Quantified data are shown as mean ± s.e.m. *p
    Emsa Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 5 article reviews
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    94
    Thermo Fisher trna
    Tau-RNA droplets form a complex coacervate phase. (A) Representative bright-field images of <t>tau-tRNA</t> droplet samples at room temperature with varying [NaCl]. (B) Droplet coverage (in %) with poly(A) or tRNA in room temperature with varying [NaCl]. Δtau187:RNA in (A) and (B) were maintained at a mass ratio of 7:1 (corresponding to a charge ratio of 1.2:1) and a total mass concentration of 2 mg/ml. (C) Droplet coverage with poly(A) or tRNA with varying Δtau187: RNA charge ratios. The total mass concentrations are indicated in the legends. Samples made by mixing of 80 μM Δtau187 with 222 μg/ml poly(A)/tRNA or 161 μM Δtau187 with 444 μg/ml tRNA gave the highest droplet coverage (%), which correspond to charge ratio of 1.3–1.2 between tau and RNA. Error bars in (B) and (C) represent the standard deviation from n = 3. (D) Representative bright-field images of tau-RNA droplets as a function of incubation temperature. To record these images, the temperature was ramped from 19ºC to 25°C at 1°C per minute to acquire confocal images with bright field illumination. The samples for these images are generated from 100 μM tau mixed with poly(U) at approximately 1:1 charge ratio in the presence of 30 mM NaCl. (E) Representative bright-field images of tau-RNA droplet samples incubated at <t>37°C</t> under otherwise different sample conditions. The concentration for tau, poly(U) RNA and NaCl are (i) 5 μM, 15 μg/ml, 0 mM; (ii) 5 μM, 15 μg/ml, 100 mM; (iii) 2.5 μM, 7.5 μg/ml, 100 mM; (iv) 1 μM, 3 μg/ml, 100 mM. Arrows highlight some of the droplets in images (ii) and (iii). Through Fig 5, scale bars are for 50 μm and all samples were prepared with Δtau187/322C in 20 mM ammonium acetate at pH 7.0. The numerical data used in (B) and (C) are included in S1 Data .
    Trna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 206 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    RPF GC content is RNase-species independent. ( A ) 3.8 A 260 homogenate from hippocampi of one P35 male mouse was digested with 100ng RNase A (Sigma, # R4875) + 60U RNase T1 (Thermo Fisher Scientific, #EN0542)/ A 260 , at 25°C for 30min and applied to a 10–50% (w/v) sucrose gradient. ( B ) 3.8 A 260 homogenate from hippocampi of one P35 mouse was digested with 5U RNase I (Ambion, #AM2294)/ A 260 , at 25°C for 45min and applied to a 10–50% (w/v) sucrose gradient. ( C ) Nucleotide composition at each position of RPFs mapped to CDS from ribosomes in (A). ( D ) Nucleotide composition at each position of RPFs mapped to CDS from ribosomes in (B). ( E ) Nucleotide composition at each position of RPFs mapped to CDS from mouse embryonic stem cells (mESCs) (data from Ingolia et al. ) ( 16 ). A 600 μl aliquot of lysate was treated with 15 μl RNase I at 100 U/μl for 45 min at 25°C. ( F ) Nucleotide composition at each position of RPFs mapped to CDS from human embryonic stem cell (hESC)-derived neurons (data from Grabole et al. ) ( 42 ). 5 U TruSeq Ribo Profile Nuclease/ A 260 at 25°C for 45 min.

    Journal: Nucleic Acids Research

    Article Title: Optimization of ribosome profiling using low-input brain tissue from fragile X syndrome model mice

    doi: 10.1093/nar/gky1292

    Figure Lengend Snippet: RPF GC content is RNase-species independent. ( A ) 3.8 A 260 homogenate from hippocampi of one P35 male mouse was digested with 100ng RNase A (Sigma, # R4875) + 60U RNase T1 (Thermo Fisher Scientific, #EN0542)/ A 260 , at 25°C for 30min and applied to a 10–50% (w/v) sucrose gradient. ( B ) 3.8 A 260 homogenate from hippocampi of one P35 mouse was digested with 5U RNase I (Ambion, #AM2294)/ A 260 , at 25°C for 45min and applied to a 10–50% (w/v) sucrose gradient. ( C ) Nucleotide composition at each position of RPFs mapped to CDS from ribosomes in (A). ( D ) Nucleotide composition at each position of RPFs mapped to CDS from ribosomes in (B). ( E ) Nucleotide composition at each position of RPFs mapped to CDS from mouse embryonic stem cells (mESCs) (data from Ingolia et al. ) ( 16 ). A 600 μl aliquot of lysate was treated with 15 μl RNase I at 100 U/μl for 45 min at 25°C. ( F ) Nucleotide composition at each position of RPFs mapped to CDS from human embryonic stem cell (hESC)-derived neurons (data from Grabole et al. ) ( 42 ). 5 U TruSeq Ribo Profile Nuclease/ A 260 at 25°C for 45 min.

    Article Snippet: The samples were digested with 100ng RNase A (Sigma, # R4875) and 60U RNase T1 (Thermo Fisher Scientific, #EN0542) per A 260 at 25°C for 30 min and stopped by chilling on ice and adding 50 U SUPERase In RNase inhibitor (Ambion, #AM2694).

    Techniques: Derivative Assay

    RPF GC content and length depend on the RNase digestion protocol. ( A ) Lysates from human iPSC neuron samples spanning a wide range of amounts were digested with 100 ng RNase A + 60U RNase T1/ A 260 at 25°C for 30 min. Monosomal RNA was extracted from monosomal fractions of sucrose gradients and quantified with Nanodrop. GC contents were calculated as in Figure 2A and the peaks of length distributions of RPFs mapped to CDS were also determined. Scatter plots with Pearson correlation coefficients show the negative correlation between 80S monosomal RNA amounts (log 2 scale) and the GC contents (black) or RPF lengths (red). ( B ) Lysates from human iPSC samples were digested with 20 ng RNase A + 12 U RNase T1/ A 260 at 25°C for 30 min. Scatter plots with Pearson correlation coefficients show the negative correlation between 80S monosomal RNA amounts (log 2 scale) and the GC contents (black) or RPF lengths (red). ( C ) Nucleotide composition at each position of RPFs mapped to CDS from mESC-derived neurons with an alternative protocol of RNase digestion (data from Zappulo et al. ) ( 43 ). 70 U RNase I at 25°C for 40 min.

    Journal: Nucleic Acids Research

    Article Title: Optimization of ribosome profiling using low-input brain tissue from fragile X syndrome model mice

    doi: 10.1093/nar/gky1292

    Figure Lengend Snippet: RPF GC content and length depend on the RNase digestion protocol. ( A ) Lysates from human iPSC neuron samples spanning a wide range of amounts were digested with 100 ng RNase A + 60U RNase T1/ A 260 at 25°C for 30 min. Monosomal RNA was extracted from monosomal fractions of sucrose gradients and quantified with Nanodrop. GC contents were calculated as in Figure 2A and the peaks of length distributions of RPFs mapped to CDS were also determined. Scatter plots with Pearson correlation coefficients show the negative correlation between 80S monosomal RNA amounts (log 2 scale) and the GC contents (black) or RPF lengths (red). ( B ) Lysates from human iPSC samples were digested with 20 ng RNase A + 12 U RNase T1/ A 260 at 25°C for 30 min. Scatter plots with Pearson correlation coefficients show the negative correlation between 80S monosomal RNA amounts (log 2 scale) and the GC contents (black) or RPF lengths (red). ( C ) Nucleotide composition at each position of RPFs mapped to CDS from mESC-derived neurons with an alternative protocol of RNase digestion (data from Zappulo et al. ) ( 43 ). 70 U RNase I at 25°C for 40 min.

    Article Snippet: The samples were digested with 100ng RNase A (Sigma, # R4875) and 60U RNase T1 (Thermo Fisher Scientific, #EN0542) per A 260 at 25°C for 30 min and stopped by chilling on ice and adding 50 U SUPERase In RNase inhibitor (Ambion, #AM2694).

    Techniques: Derivative Assay

    The GC-content correlated batch effects are caused by incomplete RNase digestion. ( A ) Hippocampi from one P35 WT mouse were homogenized and the homogenate was aliquoted for the titration experiment. 0.5 unit A 260 homogenate containing 2 μg RNA (measured with Qubit HS RNA kit) in 0.3 ml volume was used for digestion at each RNase concentration. Digested homogenates were separated on 10–50% (w/v) sucrose gradients. Profile of hippocampal ribosomes after the digestion at the lowest concentration1 [Conc.1, 4.8ng RNase A (Ambion, #AM2270) + 0.6 U RNase T1 (Thermo Fisher Scientific, #EN0542)/μg RNA × 2 μg RNA in 0.3 ml at 25°C for 30 min] and sucrose gradient fractionation. ( B ) Profile of hippocampal ribosomes after the digestion at the concentration2 (Conc.2, 24 ng RNase A + 3U RNase T1/μg RNA × 2 μg RNA in 0.3 ml at 25°C for 30 min) and sucrose gradient fractionation. ( C ) Profile of hippocampal ribosomes after the digestion at the concentration3 (Conc.3, 120 ng RNase A + 15 U RNase T1/μg RNA × 2 μg RNA in 0.3 ml at 25°C for 30 min) and sucrose gradient fractionation. ( D ) Profile of hippocampal ribosomes after the digestion at the concentration4 (Conc.4, 600 ng RNase A + 75 U RNase T1/μg RNA × 2 μg RNA in 0.3 ml at 25°C for 30 min) and sucrose gradient fractionation. ( E ) Profile of hippocampal ribosomes after the digestion at the highest concentration5 (Conc.5, 3000 ng RNase A + 375 U RNase T1/μg RNA × 2 μg RNA RNA in 0.3 ml at 25°C for 30 min) and sucrose gradient fractionation. ( F ) Scatter plots with Pearson correlation coefficients show the negative correlation between RNase concentrations (log 5 scale) and the GC contents (black) or RPF lengths (red).

    Journal: Nucleic Acids Research

    Article Title: Optimization of ribosome profiling using low-input brain tissue from fragile X syndrome model mice

    doi: 10.1093/nar/gky1292

    Figure Lengend Snippet: The GC-content correlated batch effects are caused by incomplete RNase digestion. ( A ) Hippocampi from one P35 WT mouse were homogenized and the homogenate was aliquoted for the titration experiment. 0.5 unit A 260 homogenate containing 2 μg RNA (measured with Qubit HS RNA kit) in 0.3 ml volume was used for digestion at each RNase concentration. Digested homogenates were separated on 10–50% (w/v) sucrose gradients. Profile of hippocampal ribosomes after the digestion at the lowest concentration1 [Conc.1, 4.8ng RNase A (Ambion, #AM2270) + 0.6 U RNase T1 (Thermo Fisher Scientific, #EN0542)/μg RNA × 2 μg RNA in 0.3 ml at 25°C for 30 min] and sucrose gradient fractionation. ( B ) Profile of hippocampal ribosomes after the digestion at the concentration2 (Conc.2, 24 ng RNase A + 3U RNase T1/μg RNA × 2 μg RNA in 0.3 ml at 25°C for 30 min) and sucrose gradient fractionation. ( C ) Profile of hippocampal ribosomes after the digestion at the concentration3 (Conc.3, 120 ng RNase A + 15 U RNase T1/μg RNA × 2 μg RNA in 0.3 ml at 25°C for 30 min) and sucrose gradient fractionation. ( D ) Profile of hippocampal ribosomes after the digestion at the concentration4 (Conc.4, 600 ng RNase A + 75 U RNase T1/μg RNA × 2 μg RNA in 0.3 ml at 25°C for 30 min) and sucrose gradient fractionation. ( E ) Profile of hippocampal ribosomes after the digestion at the highest concentration5 (Conc.5, 3000 ng RNase A + 375 U RNase T1/μg RNA × 2 μg RNA RNA in 0.3 ml at 25°C for 30 min) and sucrose gradient fractionation. ( F ) Scatter plots with Pearson correlation coefficients show the negative correlation between RNase concentrations (log 5 scale) and the GC contents (black) or RPF lengths (red).

    Article Snippet: The samples were digested with 100ng RNase A (Sigma, # R4875) and 60U RNase T1 (Thermo Fisher Scientific, #EN0542) per A 260 at 25°C for 30 min and stopped by chilling on ice and adding 50 U SUPERase In RNase inhibitor (Ambion, #AM2694).

    Techniques: Titration, Concentration Assay, Fractionation

    Optimization of fluorescence labeling of MAGIC Factor and MAGIC Probes. ( A ) Labeling strategy of MAGIC Factor. MAGIC Factor was fluorescently labeled with increasing amounts of Alexa Fluor 488 SDP Ester and run on a 12% SDS-PAGE gel after purification by size-exclusion chromatography. The ratios refer to dye:protein molar ratios during fluorescent labeling. The control reaction was performed with DMSO instead of the dye. The same gel was imaged for native Alexa Fluor 488 fluorescence (top gel) and after staining all protein with Sypro Orange (bottom gel). ( B ) Electrophoretic Mobility Shift Assay (EMSA) of fluorescently labeled MAGIC Factor. Proteins from A were reacted with Alexa Fluor 647-labeled dsRNA and run on a native gel to visualize the binding affinities of the proteins. The gel was visualized in the RNA channel, protein channel and FRET channel. Note that the appearance of MAGIC Factor as multiple bands is likely due to the use of an NHS-ester dye to attach Alexa Fluor 488 to the protein. Dependent on the exact location of the fluorophore molecule, each single protein molecule likely runs differently on the native polyacrylamide gel. ( C ) Quantitative assessment of the EMSA with respect to the relative shift of the dsRNA, the corrected FRET (cFRET) intensity of shifted dsRNA and the cFRET/shift ratio. ( D ) Unlabeled and fluorescently labeled MAGIC Factor were affinity purified using dsRNA-coupled agarose beads. The proteins were reacted with the beads for 1 hr, washed three times with binding buffer and then gradually eluted with increasing concentrations of KCl. At the end, remaining proteins were eluted with 1x SDS-PAGE sample buffer and all samples run on a 12% SDS-PAGE gel. As a control experiment, fluorescently labeled MAGIC Factor was reacted with agarose beads in the absence of dsRNA. Gels were imaged for native Alexa Fluor 488 fluorescence (right gel) and after staining all protein (left gel). ( E ) Effect of degree of RNA fluorescent labeling on probe hybridization kinetics. Fluorescently labeled RNA probes were gel purified to obtain one to four labeled RNA probes. They were then reacted with unlabeled sense probes to generate dsRNA in TEN 100 buffer (tris, EDTA, sodium chloride) at 95°C (positive control) or physiologic buffer resembling cytoplasmic ionic concentrations at 37°C for 30 min or 2 hr. ssRNA are shown as controls. Representative native 20% polyacrylamide gels are shown. ( F ) Quantification of RNA fluorescence intensities from ( E ). Quantified data are shown as mean ± s.e.m. *p

    Journal: eLife

    Article Title: Multiplex live single-cell transcriptional analysis demarcates cellular functional heterogeneity

    doi: 10.7554/eLife.49599

    Figure Lengend Snippet: Optimization of fluorescence labeling of MAGIC Factor and MAGIC Probes. ( A ) Labeling strategy of MAGIC Factor. MAGIC Factor was fluorescently labeled with increasing amounts of Alexa Fluor 488 SDP Ester and run on a 12% SDS-PAGE gel after purification by size-exclusion chromatography. The ratios refer to dye:protein molar ratios during fluorescent labeling. The control reaction was performed with DMSO instead of the dye. The same gel was imaged for native Alexa Fluor 488 fluorescence (top gel) and after staining all protein with Sypro Orange (bottom gel). ( B ) Electrophoretic Mobility Shift Assay (EMSA) of fluorescently labeled MAGIC Factor. Proteins from A were reacted with Alexa Fluor 647-labeled dsRNA and run on a native gel to visualize the binding affinities of the proteins. The gel was visualized in the RNA channel, protein channel and FRET channel. Note that the appearance of MAGIC Factor as multiple bands is likely due to the use of an NHS-ester dye to attach Alexa Fluor 488 to the protein. Dependent on the exact location of the fluorophore molecule, each single protein molecule likely runs differently on the native polyacrylamide gel. ( C ) Quantitative assessment of the EMSA with respect to the relative shift of the dsRNA, the corrected FRET (cFRET) intensity of shifted dsRNA and the cFRET/shift ratio. ( D ) Unlabeled and fluorescently labeled MAGIC Factor were affinity purified using dsRNA-coupled agarose beads. The proteins were reacted with the beads for 1 hr, washed three times with binding buffer and then gradually eluted with increasing concentrations of KCl. At the end, remaining proteins were eluted with 1x SDS-PAGE sample buffer and all samples run on a 12% SDS-PAGE gel. As a control experiment, fluorescently labeled MAGIC Factor was reacted with agarose beads in the absence of dsRNA. Gels were imaged for native Alexa Fluor 488 fluorescence (right gel) and after staining all protein (left gel). ( E ) Effect of degree of RNA fluorescent labeling on probe hybridization kinetics. Fluorescently labeled RNA probes were gel purified to obtain one to four labeled RNA probes. They were then reacted with unlabeled sense probes to generate dsRNA in TEN 100 buffer (tris, EDTA, sodium chloride) at 95°C (positive control) or physiologic buffer resembling cytoplasmic ionic concentrations at 37°C for 30 min or 2 hr. ssRNA are shown as controls. Representative native 20% polyacrylamide gels are shown. ( F ) Quantification of RNA fluorescence intensities from ( E ). Quantified data are shown as mean ± s.e.m. *p

    Article Snippet: Typically, 100–200 nM of RNA was reacted with MAGIC Factor in EMSA buffer (25 mM HEPES pH 7.4, 100 mM KCl, 10 mM NaCl, 0.5 mM EDTA, 1 mM TCEP, 0.1% Nonidet P-40, 5% Glycerol), 10U Superase In RNase Inhibitor (Thermo Scientific) and 0.1 mg/ml t-RNA (Sigma Aldrich) for 30 min at 4°C before loading onto a 12% native polyacrylamide gel containing 2.5% glycerol.

    Techniques: Fluorescence, Labeling, SDS Page, Purification, Size-exclusion Chromatography, Staining, Electrophoretic Mobility Shift Assay, Binding Assay, Affinity Purification, Hybridization, Positive Control

    Tau-RNA droplets form a complex coacervate phase. (A) Representative bright-field images of tau-tRNA droplet samples at room temperature with varying [NaCl]. (B) Droplet coverage (in %) with poly(A) or tRNA in room temperature with varying [NaCl]. Δtau187:RNA in (A) and (B) were maintained at a mass ratio of 7:1 (corresponding to a charge ratio of 1.2:1) and a total mass concentration of 2 mg/ml. (C) Droplet coverage with poly(A) or tRNA with varying Δtau187: RNA charge ratios. The total mass concentrations are indicated in the legends. Samples made by mixing of 80 μM Δtau187 with 222 μg/ml poly(A)/tRNA or 161 μM Δtau187 with 444 μg/ml tRNA gave the highest droplet coverage (%), which correspond to charge ratio of 1.3–1.2 between tau and RNA. Error bars in (B) and (C) represent the standard deviation from n = 3. (D) Representative bright-field images of tau-RNA droplets as a function of incubation temperature. To record these images, the temperature was ramped from 19ºC to 25°C at 1°C per minute to acquire confocal images with bright field illumination. The samples for these images are generated from 100 μM tau mixed with poly(U) at approximately 1:1 charge ratio in the presence of 30 mM NaCl. (E) Representative bright-field images of tau-RNA droplet samples incubated at 37°C under otherwise different sample conditions. The concentration for tau, poly(U) RNA and NaCl are (i) 5 μM, 15 μg/ml, 0 mM; (ii) 5 μM, 15 μg/ml, 100 mM; (iii) 2.5 μM, 7.5 μg/ml, 100 mM; (iv) 1 μM, 3 μg/ml, 100 mM. Arrows highlight some of the droplets in images (ii) and (iii). Through Fig 5, scale bars are for 50 μm and all samples were prepared with Δtau187/322C in 20 mM ammonium acetate at pH 7.0. The numerical data used in (B) and (C) are included in S1 Data .

    Journal: PLoS Biology

    Article Title: RNA stores tau reversibly in complex coacervates

    doi: 10.1371/journal.pbio.2002183

    Figure Lengend Snippet: Tau-RNA droplets form a complex coacervate phase. (A) Representative bright-field images of tau-tRNA droplet samples at room temperature with varying [NaCl]. (B) Droplet coverage (in %) with poly(A) or tRNA in room temperature with varying [NaCl]. Δtau187:RNA in (A) and (B) were maintained at a mass ratio of 7:1 (corresponding to a charge ratio of 1.2:1) and a total mass concentration of 2 mg/ml. (C) Droplet coverage with poly(A) or tRNA with varying Δtau187: RNA charge ratios. The total mass concentrations are indicated in the legends. Samples made by mixing of 80 μM Δtau187 with 222 μg/ml poly(A)/tRNA or 161 μM Δtau187 with 444 μg/ml tRNA gave the highest droplet coverage (%), which correspond to charge ratio of 1.3–1.2 between tau and RNA. Error bars in (B) and (C) represent the standard deviation from n = 3. (D) Representative bright-field images of tau-RNA droplets as a function of incubation temperature. To record these images, the temperature was ramped from 19ºC to 25°C at 1°C per minute to acquire confocal images with bright field illumination. The samples for these images are generated from 100 μM tau mixed with poly(U) at approximately 1:1 charge ratio in the presence of 30 mM NaCl. (E) Representative bright-field images of tau-RNA droplet samples incubated at 37°C under otherwise different sample conditions. The concentration for tau, poly(U) RNA and NaCl are (i) 5 μM, 15 μg/ml, 0 mM; (ii) 5 μM, 15 μg/ml, 100 mM; (iii) 2.5 μM, 7.5 μg/ml, 100 mM; (iv) 1 μM, 3 μg/ml, 100 mM. Arrows highlight some of the droplets in images (ii) and (iii). Through Fig 5, scale bars are for 50 μm and all samples were prepared with Δtau187/322C in 20 mM ammonium acetate at pH 7.0. The numerical data used in (B) and (C) are included in S1 Data .

    Article Snippet: For the gel shift assay, protein was incubated with tRNA at 37°C for 10 minutes in the presence of 0.5 mM EDTA, 0.5 mM MgCl2, 2 U SUPERase• In RNase Inhibitor (Thermo Fisher, Waltham, MA), 0.01% IGEPAL CA-630 (Sigma-Aldrich), and then applied to a TBE 8% Polyacrylamide Gel (Thermo Fisher, Waltham, MA).

    Techniques: Concentration Assay, Standard Deviation, Incubation, Generated