murine rnase inhibitor  (New England Biolabs)


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    RNase Inhibitor Murine
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    RNase Inhibitor Murine 15 000 units
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    m0314l
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    Enzyme Inhibitors
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    New England Biolabs murine rnase inhibitor
    RNase Inhibitor Murine
    RNase Inhibitor Murine 15 000 units
    https://www.bioz.com/result/murine rnase inhibitor/product/New England Biolabs
    Average 95 stars, based on 59 article reviews
    Price from $9.99 to $1999.99
    murine rnase inhibitor - by Bioz Stars, 2020-02
    95/100 stars

    Images

    1) Product Images from "RNA targeting with CRISPR-Cas13a"

    Article Title: RNA targeting with CRISPR-Cas13a

    Journal: Nature

    doi: 10.1038/nature24049

    Biochemical characterization of LwaCas13a RNA cleavage activity a, LwaCas13a has more active RNAse activity than LshCas13a. b, Gel electrophoresis of ssRNA1 after incubation with LwaCas13a and with and without crRNA 1 for varying amounts of times. c, Gel electrophoresis of ssRNA1 after incubation with varying amounts of LwaCas13a-crRNA complex. d, Sequence and structure of ssRNA 4 and ssRNA 5. crRNA spacer sequence is highlighted in blue. e, Gel electrophoresis of ssRNA 4 and ssRNA 5 after incubation with LwaCas13a and crRNA 1. f, Sequence and structure of ssRNA 4 with sites of poly-x modifications highlighted in red. crRNA spacer sequence is highlighted in blue. g, Gel electrophoresis of ssRNA 4 with each of 4 possible poly-x modifications incubated with LwaCas13a and crRNA 1. h, LwaCas13a can process pre-crRNA from the L. wadei CRISPR-Cas locus. i, Cleavage efficiency of ssRNA 1 for crRNA spacer truncations after incubation with LwaCas13a.
    Figure Legend Snippet: Biochemical characterization of LwaCas13a RNA cleavage activity a, LwaCas13a has more active RNAse activity than LshCas13a. b, Gel electrophoresis of ssRNA1 after incubation with LwaCas13a and with and without crRNA 1 for varying amounts of times. c, Gel electrophoresis of ssRNA1 after incubation with varying amounts of LwaCas13a-crRNA complex. d, Sequence and structure of ssRNA 4 and ssRNA 5. crRNA spacer sequence is highlighted in blue. e, Gel electrophoresis of ssRNA 4 and ssRNA 5 after incubation with LwaCas13a and crRNA 1. f, Sequence and structure of ssRNA 4 with sites of poly-x modifications highlighted in red. crRNA spacer sequence is highlighted in blue. g, Gel electrophoresis of ssRNA 4 with each of 4 possible poly-x modifications incubated with LwaCas13a and crRNA 1. h, LwaCas13a can process pre-crRNA from the L. wadei CRISPR-Cas locus. i, Cleavage efficiency of ssRNA 1 for crRNA spacer truncations after incubation with LwaCas13a.

    Techniques Used: Activity Assay, Nucleic Acid Electrophoresis, Incubation, Sequencing, CRISPR

    2) Product Images from "eIF3d is an mRNA cap-binding protein required for specialized translation initiation"

    Article Title: eIF3d is an mRNA cap-binding protein required for specialized translation initiation

    Journal: Nature

    doi: 10.1038/nature18954

    eIF3d cap-binding activity is required for efficient 48S initiation complex formation on specific mRNAs a , Phosphorimage of SDS gel resolving RNase-protected 32 P-cap-labeled c-Jun 5′ UTR RNA crosslinked to eIF3 in the presence of competitor ligands. b , Electrostatic surface view of the eIF3d cap-binding domain colored by charge, with a zoomed view of single stranded RNA (ssRNA) and cap analog modeled according to their positions bound to DXO 15 . Positive charge is colored blue and negative charge is in red, and the RNA gate is removed for clarity. c , Phosphorimage of SDS gel resolving RNase-protected 32 P-cap-labeled c-Jun 5′ UTR RNA crosslinked to wild type or helix α5 or helix α11-mutant eIF3. eIF3d-helix α5 mutant (D249Q/V262I/Y263A), helix α11 mutant (T317E/N320E/H321A). WT, wild type. d , Incorporation of c-Jun and ACTB mRNA into initiation complexes by wild type, helix α5, or helix α11-mutant eIF3d as measured by quantitative RT-PCR. mRNA-ribosome association is expressed as the ratio between the quantity of mRNA transcripts to 18S rRNA and normalized to the wild type sample. The results are representative of three independent experiments and given as the mean ± s.d. from a representative quantitative RT-PCR experiment performed in duplicate.
    Figure Legend Snippet: eIF3d cap-binding activity is required for efficient 48S initiation complex formation on specific mRNAs a , Phosphorimage of SDS gel resolving RNase-protected 32 P-cap-labeled c-Jun 5′ UTR RNA crosslinked to eIF3 in the presence of competitor ligands. b , Electrostatic surface view of the eIF3d cap-binding domain colored by charge, with a zoomed view of single stranded RNA (ssRNA) and cap analog modeled according to their positions bound to DXO 15 . Positive charge is colored blue and negative charge is in red, and the RNA gate is removed for clarity. c , Phosphorimage of SDS gel resolving RNase-protected 32 P-cap-labeled c-Jun 5′ UTR RNA crosslinked to wild type or helix α5 or helix α11-mutant eIF3. eIF3d-helix α5 mutant (D249Q/V262I/Y263A), helix α11 mutant (T317E/N320E/H321A). WT, wild type. d , Incorporation of c-Jun and ACTB mRNA into initiation complexes by wild type, helix α5, or helix α11-mutant eIF3d as measured by quantitative RT-PCR. mRNA-ribosome association is expressed as the ratio between the quantity of mRNA transcripts to 18S rRNA and normalized to the wild type sample. The results are representative of three independent experiments and given as the mean ± s.d. from a representative quantitative RT-PCR experiment performed in duplicate.

    Techniques Used: Binding Assay, Activity Assay, SDS-Gel, Labeling, Mutagenesis, Quantitative RT-PCR

    eIF4E recognizes the 5′ end of the c-Jun mRNA less efficiently than ACTB mRNA a , Coomassie blue stained SDS gel of recombinant human eIF4E expressed in E. coli. b , Phosphorimage of SDS gel resolving RNase-protected 32 P-cap-labeled ACTB or c-Jun 5′ UTR RNA crosslinked to eIF4E. The result is representative of three independent experiments. For gel source data, see Supplementary Figure 1 .
    Figure Legend Snippet: eIF4E recognizes the 5′ end of the c-Jun mRNA less efficiently than ACTB mRNA a , Coomassie blue stained SDS gel of recombinant human eIF4E expressed in E. coli. b , Phosphorimage of SDS gel resolving RNase-protected 32 P-cap-labeled ACTB or c-Jun 5′ UTR RNA crosslinked to eIF4E. The result is representative of three independent experiments. For gel source data, see Supplementary Figure 1 .

    Techniques Used: Staining, SDS-Gel, Recombinant, Labeling

    5' end recognition of c-Jun mRNA is eIF4F-independent a , Distribution of c-Jun or ACTB mRNA-containing initiation complexes in programmed 293T cell in vitro translation extracts. The mRNA abundance (black line) is expressed as the fraction of total recovered transcripts. The results are given as the mean ± standard deviation (s.d.) of a representative quantitative RT-PCR experiment performed in duplicate. The polysome profile (gray line) is plotted as relative absorbance at 254 nm versus elution fractions. b , Western blot analysis of initiation factors in 48S translation complexes formed on c-Jun and ACTB mRNAs. 293T, total protein from 293T in vitro translation extracts. For gel source data, see Supplementary Figure 1 . c , Phosphorimage of SDS gel resolving RNase-protected 32 P-internal or 32 P-cap-labeled c-Jun 5' UTR RNA crosslinked to eIF3 subunits. Recombinant eIF3a migrates at ~100 kDa due to a C-terminal truncation 26 . The results of a - c are representative of three independent experiments.
    Figure Legend Snippet: 5' end recognition of c-Jun mRNA is eIF4F-independent a , Distribution of c-Jun or ACTB mRNA-containing initiation complexes in programmed 293T cell in vitro translation extracts. The mRNA abundance (black line) is expressed as the fraction of total recovered transcripts. The results are given as the mean ± standard deviation (s.d.) of a representative quantitative RT-PCR experiment performed in duplicate. The polysome profile (gray line) is plotted as relative absorbance at 254 nm versus elution fractions. b , Western blot analysis of initiation factors in 48S translation complexes formed on c-Jun and ACTB mRNAs. 293T, total protein from 293T in vitro translation extracts. For gel source data, see Supplementary Figure 1 . c , Phosphorimage of SDS gel resolving RNase-protected 32 P-internal or 32 P-cap-labeled c-Jun 5' UTR RNA crosslinked to eIF3 subunits. Recombinant eIF3a migrates at ~100 kDa due to a C-terminal truncation 26 . The results of a - c are representative of three independent experiments.

    Techniques Used: In Vitro, Standard Deviation, Quantitative RT-PCR, Western Blot, SDS-Gel, Labeling, Recombinant

    3) Product Images from "RNA binding to CBP stimulates histone acetylation and transcription"

    Article Title: RNA binding to CBP stimulates histone acetylation and transcription

    Journal: Cell

    doi: 10.1016/j.cell.2016.12.020

    CBP interacts with RNA in vivo A) Native RNA-IP of CBP. Top, RNA immunprecipitated with CBP. Bottom, CBP western blot. B) PAR-CLIP protocol. 4-Thiouridine (4-SU). C) CBP PAR-CLIP required 4-SU: top, autoradiography; bottom, CBP western blot.. D) Quantification of CBP PAR-CLIP. Error bars represent mean +/− s.e.m; n=4. E) CBP PAR-CLIP signal was sensitive to RNAse. 1× RNAse cocktail contained: RNAse A (0.01mU/ul) + RNase T1 (0.4mU/ul). F) Quantification of RNase titration. Error bars represent mean +/− s.e.m; n=4; P -values from two-tailed Student’s t-test: *P
    Figure Legend Snippet: CBP interacts with RNA in vivo A) Native RNA-IP of CBP. Top, RNA immunprecipitated with CBP. Bottom, CBP western blot. B) PAR-CLIP protocol. 4-Thiouridine (4-SU). C) CBP PAR-CLIP required 4-SU: top, autoradiography; bottom, CBP western blot.. D) Quantification of CBP PAR-CLIP. Error bars represent mean +/− s.e.m; n=4. E) CBP PAR-CLIP signal was sensitive to RNAse. 1× RNAse cocktail contained: RNAse A (0.01mU/ul) + RNase T1 (0.4mU/ul). F) Quantification of RNase titration. Error bars represent mean +/− s.e.m; n=4; P -values from two-tailed Student’s t-test: *P

    Techniques Used: In Vivo, Western Blot, Cross-linking Immunoprecipitation, Autoradiography, Titration, Two Tailed Test

    4) Product Images from "RNA binding to CBP stimulates histone acetylation and transcription"

    Article Title: RNA binding to CBP stimulates histone acetylation and transcription

    Journal: Cell

    doi: 10.1016/j.cell.2016.12.020

    CBP interacts with RNA in vivo A) Native RNA-IP of CBP. Top, RNA immunprecipitated with CBP. Bottom, CBP western blot. B) PAR-CLIP protocol. 4-Thiouridine (4-SU). C) CBP PAR-CLIP required 4-SU: top, autoradiography; bottom, CBP western blot.. D) Quantification of CBP PAR-CLIP. Error bars represent mean +/− s.e.m; n=4. E) CBP PAR-CLIP signal was sensitive to RNAse. 1× RNAse cocktail contained: RNAse A (0.01mU/ul) + RNase T1 (0.4mU/ul). F) Quantification of RNase titration. Error bars represent mean +/− s.e.m; n=4; P -values from two-tailed Student’s t-test: *P
    Figure Legend Snippet: CBP interacts with RNA in vivo A) Native RNA-IP of CBP. Top, RNA immunprecipitated with CBP. Bottom, CBP western blot. B) PAR-CLIP protocol. 4-Thiouridine (4-SU). C) CBP PAR-CLIP required 4-SU: top, autoradiography; bottom, CBP western blot.. D) Quantification of CBP PAR-CLIP. Error bars represent mean +/− s.e.m; n=4. E) CBP PAR-CLIP signal was sensitive to RNAse. 1× RNAse cocktail contained: RNAse A (0.01mU/ul) + RNase T1 (0.4mU/ul). F) Quantification of RNase titration. Error bars represent mean +/− s.e.m; n=4; P -values from two-tailed Student’s t-test: *P

    Techniques Used: In Vivo, Western Blot, Cross-linking Immunoprecipitation, Autoradiography, Titration, Two Tailed Test

    5) Product Images from "Polyvinylsulfonic acid: A Low-cost RNase inhibitor for enhanced RNA preservation and cell-free protein translation"

    Article Title: Polyvinylsulfonic acid: A Low-cost RNase inhibitor for enhanced RNA preservation and cell-free protein translation

    Journal: Bioengineered

    doi: 10.1080/21655979.2017.1313648

    Inhibition of RNase Activity with PVSA. The relative RNase Activity of both RNase A and E. coli lysate was measured at varying concentrations of PVSA using RNaseAlert® (Ambion). The amount of PVSA required for 50% inhibition (IC 50 , inset) was determined from normalized data fit to a reciprocal semi-log response curve (n = 3, error bars represent 1 standard deviation).
    Figure Legend Snippet: Inhibition of RNase Activity with PVSA. The relative RNase Activity of both RNase A and E. coli lysate was measured at varying concentrations of PVSA using RNaseAlert® (Ambion). The amount of PVSA required for 50% inhibition (IC 50 , inset) was determined from normalized data fit to a reciprocal semi-log response curve (n = 3, error bars represent 1 standard deviation).

    Techniques Used: Inhibition, Activity Assay, Standard Deviation

    6) Product Images from "nextPARS: parallel probing of RNA structures in Illumina"

    Article Title: nextPARS: parallel probing of RNA structures in Illumina

    Journal: RNA

    doi: 10.1261/rna.063073.117

    Summary of the different steps performed in the nextPARS protocol. From the cells or tissue of interest ( A ), total RNA is extracted ( B ) and then poly(A) + RNA is selected ( C ) to initially prepare the samples for nextPARS analyses. Once the quality and quantity of poly(A) + RNA samples is confirmed, RNA samples are denatured and in vitro folded to perform the enzymatic probing of the molecules with the corresponding concentrations of RNase V1 and S1 nuclease ( D ). For the library preparation using the Illumina TruSeq Small RNA Sample Preparation Kit, an initial phosphatase treatment of the 3′ends and a kinase treatment of the 5′ ends are required ( E ) to then ligate the corresponding 5′ and 3′ adapters at the ends of the RNA fragments ( F ). Then a reverse transcription of the RNA fragments and a PCR amplification are performed to obtain the library ( G ). The library is size-selected to get rid of primers and adapters dimers using an acrylamide gel and a final quality control is performed ( H ). Libraries are sequenced in single-reads with read lengths of 50 nucleotides (nt) using Illumina sequencing platforms ( I ) and computational analyses are done as described in the Materials and Methods section in order to map Illumina reads and determine the enzymatic cleavage points, using the first nucleotide in the 5′ end of the reads (which correspond to the 5′end of original RNA fragments) ( J ).
    Figure Legend Snippet: Summary of the different steps performed in the nextPARS protocol. From the cells or tissue of interest ( A ), total RNA is extracted ( B ) and then poly(A) + RNA is selected ( C ) to initially prepare the samples for nextPARS analyses. Once the quality and quantity of poly(A) + RNA samples is confirmed, RNA samples are denatured and in vitro folded to perform the enzymatic probing of the molecules with the corresponding concentrations of RNase V1 and S1 nuclease ( D ). For the library preparation using the Illumina TruSeq Small RNA Sample Preparation Kit, an initial phosphatase treatment of the 3′ends and a kinase treatment of the 5′ ends are required ( E ) to then ligate the corresponding 5′ and 3′ adapters at the ends of the RNA fragments ( F ). Then a reverse transcription of the RNA fragments and a PCR amplification are performed to obtain the library ( G ). The library is size-selected to get rid of primers and adapters dimers using an acrylamide gel and a final quality control is performed ( H ). Libraries are sequenced in single-reads with read lengths of 50 nucleotides (nt) using Illumina sequencing platforms ( I ) and computational analyses are done as described in the Materials and Methods section in order to map Illumina reads and determine the enzymatic cleavage points, using the first nucleotide in the 5′ end of the reads (which correspond to the 5′end of original RNA fragments) ( J ).

    Techniques Used: In Vitro, Sample Prep, Polymerase Chain Reaction, Amplification, Acrylamide Gel Assay, Sequencing

    Probing of RNA molecules with RNase A enzyme. Examples of the signals obtained in some RNA molecules when performing nextPARS using RNase A, an enzyme that cuts specifically in single-stranded cytosines (C) and uracils (U). Scores were calculated for each site by first capping all read counts for a given transcript at the 95th percentile and then normalizing to have a maximum of 1 (as done in the “Computation of nextPARS scores” of the Materials and Methods, but since Rnase A is the only enzyme in this case, there will be no subtraction performed, so all values will then fall in the range of 0 to 1). Cuts are considered for signals above a threshold of 0.8. ( A ]). In green, nucleotides with a cut signal above 0.8; green crosses (+) show cuts obtained in a C or U; pink asterisks (*) show cuts obtained in a G or A; and blue arrows (→) show cuts obtained in double-stranded positions. ( B ) Table summarizing the total number (N) and percentages (%) of cuts with a signal above 0.8 threshold obtained in five different RNA fragments with known secondary structure (TETp4p6, TETp9-9.1, SRA, B2, U1): first column, N and % of cuts with a signal above 0.8 in the molecules; second column, N and % of these cuts in C or U nucleotides; and third column, N and % of cuts in G or A nucleotides.
    Figure Legend Snippet: Probing of RNA molecules with RNase A enzyme. Examples of the signals obtained in some RNA molecules when performing nextPARS using RNase A, an enzyme that cuts specifically in single-stranded cytosines (C) and uracils (U). Scores were calculated for each site by first capping all read counts for a given transcript at the 95th percentile and then normalizing to have a maximum of 1 (as done in the “Computation of nextPARS scores” of the Materials and Methods, but since Rnase A is the only enzyme in this case, there will be no subtraction performed, so all values will then fall in the range of 0 to 1). Cuts are considered for signals above a threshold of 0.8. ( A ]). In green, nucleotides with a cut signal above 0.8; green crosses (+) show cuts obtained in a C or U; pink asterisks (*) show cuts obtained in a G or A; and blue arrows (→) show cuts obtained in double-stranded positions. ( B ) Table summarizing the total number (N) and percentages (%) of cuts with a signal above 0.8 threshold obtained in five different RNA fragments with known secondary structure (TETp4p6, TETp9-9.1, SRA, B2, U1): first column, N and % of cuts with a signal above 0.8 in the molecules; second column, N and % of these cuts in C or U nucleotides; and third column, N and % of cuts in G or A nucleotides.

    Techniques Used:

    7) Product Images from "Transcriptome-Wide Analyses of 5?-Ends in RNase J Mutants of a Gram-Positive Pathogen Reveal a Role in RNA Maturation, Regulation and Degradation"

    Article Title: Transcriptome-Wide Analyses of 5?-Ends in RNase J Mutants of a Gram-Positive Pathogen Reveal a Role in RNA Maturation, Regulation and Degradation

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1004207

    Both maturation and inactivation of RNase P RNA is carried out by RNase J. A) Histogram showing the percentage of reads mapping to a given position, out of the total number of reads mapping to the putative SA1279- rnpB operon in each strain (shown in parentheses). Only positions of interest are included, but the full data-set can be found in Table S6 . +1: The putative transcription start site of SA1279. +452 to +477: The RNase J mutants accumulate RNA with 5′-ends in this region. +485: The putative transcription start site of rnpB , a major detected RNA species in the WT, ΔY and ΔcshA, but very reduced in the RNase J mutants. +499: A major detected RNA species in the WT, ΔY and ΔcshA, however it is absent from the RNase J1 mutants and reduced in the ΔJ2 strain. B) The layout of the region around SA1279 and rnpB . DNA is represented as a wavy line, and RNA transcripts as straight black lines. (PP)P indicates a mix of tri- and mono-phosphorylated RNA, generated by pyrophosphohydrolases. Small blue arrows indicate the PCR-primers used to amplify circularised RnpB and SA1279-RnpB for mapping the 5′ and 3′-ends. R1 indicates the probe used for the Northern blot shown in Figure S2 . C) A blow-up of the region from +420 to +540, showing the proposed model for converting the +1 transcript into mature RnpB. P indicates mono-phosphorylation. D) Predicted secondary structures of RnpB, generated using mfold with default settings [30] , and based on the crystal structures of RNase P RNA [27] , [31] . Within the RNase P structure, the thin dotted arrows indicate the path of the RNA through the secondary and tertiary structure of RNase P, the RBS and start codon of SA1278 are in bold, and the region where the anti-sense RNA can hybridise is indicated with a thick black line. E) The difference in average length of RnpB in WT, ΔJ1, and ΔJ1ΔJ2 strains, revealed by the length of the PCR-product generated across the 5′/3′ junction. Results of the cloned and sequenced PCR-products are shown in Table 6 . M: Marker.
    Figure Legend Snippet: Both maturation and inactivation of RNase P RNA is carried out by RNase J. A) Histogram showing the percentage of reads mapping to a given position, out of the total number of reads mapping to the putative SA1279- rnpB operon in each strain (shown in parentheses). Only positions of interest are included, but the full data-set can be found in Table S6 . +1: The putative transcription start site of SA1279. +452 to +477: The RNase J mutants accumulate RNA with 5′-ends in this region. +485: The putative transcription start site of rnpB , a major detected RNA species in the WT, ΔY and ΔcshA, but very reduced in the RNase J mutants. +499: A major detected RNA species in the WT, ΔY and ΔcshA, however it is absent from the RNase J1 mutants and reduced in the ΔJ2 strain. B) The layout of the region around SA1279 and rnpB . DNA is represented as a wavy line, and RNA transcripts as straight black lines. (PP)P indicates a mix of tri- and mono-phosphorylated RNA, generated by pyrophosphohydrolases. Small blue arrows indicate the PCR-primers used to amplify circularised RnpB and SA1279-RnpB for mapping the 5′ and 3′-ends. R1 indicates the probe used for the Northern blot shown in Figure S2 . C) A blow-up of the region from +420 to +540, showing the proposed model for converting the +1 transcript into mature RnpB. P indicates mono-phosphorylation. D) Predicted secondary structures of RnpB, generated using mfold with default settings [30] , and based on the crystal structures of RNase P RNA [27] , [31] . Within the RNase P structure, the thin dotted arrows indicate the path of the RNA through the secondary and tertiary structure of RNase P, the RBS and start codon of SA1278 are in bold, and the region where the anti-sense RNA can hybridise is indicated with a thick black line. E) The difference in average length of RnpB in WT, ΔJ1, and ΔJ1ΔJ2 strains, revealed by the length of the PCR-product generated across the 5′/3′ junction. Results of the cloned and sequenced PCR-products are shown in Table 6 . M: Marker.

    Techniques Used: Generated, Polymerase Chain Reaction, Northern Blot, Clone Assay, Marker

    SA1075 mRNA inactivation by RNase J competes with translation initiation. A) Histogram showing the percentage of reads mapping to a given position, out of the total number of reads mapping to the SA1075 transcript in each strain (shown in parentheses). Only positions of interest are included, but the full data-set can be found in Table S8 . +1: The putative transcription start site. +16: The major detected RNA species in the WT, ΔY and ΔcshA. +45: Position where ΔJ1ΔJ2 differs from ΔJ1, ΔJ2 and J1 AGA , and appears similar to WT. B) Important positions indicated on the SA1075 gene. +1: Transcription Start Site. RBS: Ribosome Binding Site. R2 indicates the probe used for the Northern blot in panel D. C) Hairpin structure predicted by the mfold algorithm, which sequesters the RBS and start-codon (shown in bold). No secondary structure was predicted for the 50 nucleotides downstream of position +45. D) Northern blot of the SA1075 transcript, using probe R2. The +1 and +16 RNA species are not resolved, and can be seen as a single band, however the +45 species is clearly visible in the ΔJ1, ΔJ2, and J1 AGA strains. The marker was stained with methylene blue and photographed. As a loading control, the Northern blot was stripped and re-probed to detect the 5S rRNA. E) The proposed model for determining the fate of SA1075 mRNA via competition between RNase J, ribosomes and the nuclease that cleaves at position +16. (PP)P indicates a mix of tri- and mono-phosphorylated RNA, generated by pyrophosphohydrolases. Nascent SA1075 mRNA can either be occupied by ribosomes, binding to the RBS, or form Hairpin I which sequesters the RBS. Ribosomes will shield position +45 from RNase J, but the hairpin will not. If cleavage at position +16 occurs before RNase J has cleaved at position +45, then the RBS will be liberated from Hairpin I, and ribosomes can initiate translation. If ever the +45 cut is made by RNase J, then the mRNA, which no longer has RBS or start-codon, is immediately degraded (possibly by the RNase J1+J2 complex). Either RNase J1 or RNase J2 can perform a cleavage at position +45. The loss of both RNases prevents the +45 RNA species from being generated, thus explaining why the WT and the ΔJ1ΔJ2 strains appear similar in panel A (see discussion for details and other potential explanations).
    Figure Legend Snippet: SA1075 mRNA inactivation by RNase J competes with translation initiation. A) Histogram showing the percentage of reads mapping to a given position, out of the total number of reads mapping to the SA1075 transcript in each strain (shown in parentheses). Only positions of interest are included, but the full data-set can be found in Table S8 . +1: The putative transcription start site. +16: The major detected RNA species in the WT, ΔY and ΔcshA. +45: Position where ΔJ1ΔJ2 differs from ΔJ1, ΔJ2 and J1 AGA , and appears similar to WT. B) Important positions indicated on the SA1075 gene. +1: Transcription Start Site. RBS: Ribosome Binding Site. R2 indicates the probe used for the Northern blot in panel D. C) Hairpin structure predicted by the mfold algorithm, which sequesters the RBS and start-codon (shown in bold). No secondary structure was predicted for the 50 nucleotides downstream of position +45. D) Northern blot of the SA1075 transcript, using probe R2. The +1 and +16 RNA species are not resolved, and can be seen as a single band, however the +45 species is clearly visible in the ΔJ1, ΔJ2, and J1 AGA strains. The marker was stained with methylene blue and photographed. As a loading control, the Northern blot was stripped and re-probed to detect the 5S rRNA. E) The proposed model for determining the fate of SA1075 mRNA via competition between RNase J, ribosomes and the nuclease that cleaves at position +16. (PP)P indicates a mix of tri- and mono-phosphorylated RNA, generated by pyrophosphohydrolases. Nascent SA1075 mRNA can either be occupied by ribosomes, binding to the RBS, or form Hairpin I which sequesters the RBS. Ribosomes will shield position +45 from RNase J, but the hairpin will not. If cleavage at position +16 occurs before RNase J has cleaved at position +45, then the RBS will be liberated from Hairpin I, and ribosomes can initiate translation. If ever the +45 cut is made by RNase J, then the mRNA, which no longer has RBS or start-codon, is immediately degraded (possibly by the RNase J1+J2 complex). Either RNase J1 or RNase J2 can perform a cleavage at position +45. The loss of both RNases prevents the +45 RNA species from being generated, thus explaining why the WT and the ΔJ1ΔJ2 strains appear similar in panel A (see discussion for details and other potential explanations).

    Techniques Used: Binding Assay, Northern Blot, Marker, Staining, Generated

    mRNA maturation by RNase J reveals a potential regulation of translation. A) Histogram showing the percentage of reads mapping to a given position, out of the total number of reads mapping to the SA2322 transcript in each strain (shown in parentheses). Only positions of interest are included, but the full data-set can be found in Table S7 . +1 and +2: The putative transcription start sites. +52: A major detected RNA species in the WT, ΔY and ΔcshA, however it is absent from the RNase J1 mutants and strongly reduced in the ΔJ2 strain. B) The SA2322 locus with important positions indicated. C) A schematic view of the fate of SA2322 transcripts. A newly formed transcript can form a secondary structure, shown in panel D, which partially sequesters the ribosome binding site (RBS). RNase J can shorten the transcript by 51 nt, and is presumably blocked from further exonucleolytic digestion by ribosomes binding to the RBS. (PP)P indicates a mix of tri- and mono-phosphorylated RNA, generated by pyrophosphohydrolases. D) Predicted secondary structure at the 5′-end of the SA2322 transcript. ΔG values predicted by the mfold algorithm are in kcal/mol. RBS and start codon are indicated in bold.
    Figure Legend Snippet: mRNA maturation by RNase J reveals a potential regulation of translation. A) Histogram showing the percentage of reads mapping to a given position, out of the total number of reads mapping to the SA2322 transcript in each strain (shown in parentheses). Only positions of interest are included, but the full data-set can be found in Table S7 . +1 and +2: The putative transcription start sites. +52: A major detected RNA species in the WT, ΔY and ΔcshA, however it is absent from the RNase J1 mutants and strongly reduced in the ΔJ2 strain. B) The SA2322 locus with important positions indicated. C) A schematic view of the fate of SA2322 transcripts. A newly formed transcript can form a secondary structure, shown in panel D, which partially sequesters the ribosome binding site (RBS). RNase J can shorten the transcript by 51 nt, and is presumably blocked from further exonucleolytic digestion by ribosomes binding to the RBS. (PP)P indicates a mix of tri- and mono-phosphorylated RNA, generated by pyrophosphohydrolases. D) Predicted secondary structure at the 5′-end of the SA2322 transcript. ΔG values predicted by the mfold algorithm are in kcal/mol. RBS and start codon are indicated in bold.

    Techniques Used: Binding Assay, Generated

    8) Product Images from "A long non-coding RNA is required for targeting centromeric protein A to the human centromere"

    Article Title: A long non-coding RNA is required for targeting centromeric protein A to the human centromere

    Journal: eLife

    doi: 10.7554/eLife.03254

    Centromeric transcripts are 1.3 kb in length. ( A ) To determine the size of the centromeric α-satellite transcripts, the graph of the distance (in y) between the border of the Northern blot and each band of the molecular weight as a function of the number of bases was made. The distance of the centromeric α-satellite transcript band was analyzed using the standard curve from this graph to deduce its size. ( B ) Total RNAs treated with RNase A were separated on a denaturing gel, and revealed by Northern blot with radiolabeled centromeric α-satellite probes. ( C ) eG1-synchronized cells were treated, or not, with α-amanitin (2 hr). RNAs were processed and analyzed on Northern blot as in ( B ) to examine whether trace DNA contamination could yield the same band as in ( A ). DOI: http://dx.doi.org/10.7554/eLife.03254.010
    Figure Legend Snippet: Centromeric transcripts are 1.3 kb in length. ( A ) To determine the size of the centromeric α-satellite transcripts, the graph of the distance (in y) between the border of the Northern blot and each band of the molecular weight as a function of the number of bases was made. The distance of the centromeric α-satellite transcript band was analyzed using the standard curve from this graph to deduce its size. ( B ) Total RNAs treated with RNase A were separated on a denaturing gel, and revealed by Northern blot with radiolabeled centromeric α-satellite probes. ( C ) eG1-synchronized cells were treated, or not, with α-amanitin (2 hr). RNAs were processed and analyzed on Northern blot as in ( B ) to examine whether trace DNA contamination could yield the same band as in ( A ). DOI: http://dx.doi.org/10.7554/eLife.03254.010

    Techniques Used: Northern Blot, Molecular Weight

    9) Product Images from "High-resolution structure of Cas13b and biochemical characterization of RNA targeting and cleavage"

    Article Title: High-resolution structure of Cas13b and biochemical characterization of RNA targeting and cleavage

    Journal: Cell reports

    doi: 10.1016/j.celrep.2019.02.094

    PbuCas13b crRNA recognition and processing. A. Diagram of crRNA substrate co-crystallized with PbuCas13b. Direct repeat nucleotides are colored red and spacer nucleotides in light blue (full spacer is not shown). Watson-Crick base pairing denoted by black lines; non-Watson-Crick base pairing denoted by gray lines. B. The structure of crRNA within the crystallized PbuCas13b complex. The coloring is consistent with panel (A) and individual bases are numbered (−1 to −36 in the crRNA, 1 for spacer). C. Model of the 3′ end of the crRNA showing the catalytic residue K393 of the crRNA processing site and additional PbuCas13b residues that coordinate the crRNA. D. Analysis of Lid domain residues predicted to coordinate and process crRNA within PbuCas13b. Right, schematic shows Cas13b-mediated RNA degradation. The upper panel shows collateral RNase activity in FLUORESCENT COLLATERAL RNA–CLEAVAGE assays with Lid domain mutants (asterisk indicates nearly undetectable levels of fluorescence); lower panel shows processing of crRNA by these mutants. Cartoons of the expected cleavage products are shown to the left of the gel; cleavage bands and expected sizes indicated by red triangles to the right of the gel.
    Figure Legend Snippet: PbuCas13b crRNA recognition and processing. A. Diagram of crRNA substrate co-crystallized with PbuCas13b. Direct repeat nucleotides are colored red and spacer nucleotides in light blue (full spacer is not shown). Watson-Crick base pairing denoted by black lines; non-Watson-Crick base pairing denoted by gray lines. B. The structure of crRNA within the crystallized PbuCas13b complex. The coloring is consistent with panel (A) and individual bases are numbered (−1 to −36 in the crRNA, 1 for spacer). C. Model of the 3′ end of the crRNA showing the catalytic residue K393 of the crRNA processing site and additional PbuCas13b residues that coordinate the crRNA. D. Analysis of Lid domain residues predicted to coordinate and process crRNA within PbuCas13b. Right, schematic shows Cas13b-mediated RNA degradation. The upper panel shows collateral RNase activity in FLUORESCENT COLLATERAL RNA–CLEAVAGE assays with Lid domain mutants (asterisk indicates nearly undetectable levels of fluorescence); lower panel shows processing of crRNA by these mutants. Cartoons of the expected cleavage products are shown to the left of the gel; cleavage bands and expected sizes indicated by red triangles to the right of the gel.

    Techniques Used: Activity Assay, Fluorescence

    Related Articles

    Clone Assay:

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    Article Snippet: In vitro transcription assay Transcription reactions were in 25 μl total volume containing 25 mM tris-HCl (pH 8.0), 10 mM MgCl2 , 64 mM NaCl, BSA (100 μg/ml), 1 mM dithiothreitol (DTT), 400 μM ATP, 150 μM guanosine-5′-triphosphate (GTP), 150 μM cytidine-5′-triphosphate (CTP), 10 μM uridine 5′-triphosphate (UTP), 0.02 μM [α-32 P]UTP, and 4 U RNase Inhibitor Murine (New England Biolabs). .. The transcription template was added at 4 nM and consisted of a restriction cut (Hind III/Eco RI), purified (QIAquick PCR Purification Kit) human LSP/HSP plasmid, where a fragment consisting of positions 325 to 742 of human mtDNA was cloned between the Sma I and Hind III sites of pUC18 ( ).

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    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
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    Centrifugation:

    Article Title: Ubiquitination-dependent control of sexual differentiation in fission yeast
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    Article Title: A length-dependent evolutionarily conserved pathway controls nuclear export of circular RNAs
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    Amplification:

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: .. For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein. .. Expression was verified by separating 2.5 μl of the reaction on a Novex 10–20% Tris–glycine gel (Thermo Fisher Scientific).

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: .. For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein. .. Expression was verified by separating 2.5 μl of the reaction on a Novex 10–20% Tris–glycine gel (Thermo Fisher Scientific).

    Autoradiography:

    Article Title: Impaired spliceosomal UsnRNP assembly leads to Sm mRNA down-regulation and Sm protein degradation
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    Construct:

    Article Title: 3′ end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character—RNA-Seq analyses
    Article Snippet: Most transcription reactions were carried out with constructs in which the nontemplate strand extends only to position +2, a common practice in the field ( , ). .. RNase Inhibitor Murine (New England BioLabs) was added to each transcription mixture before and after the reaction, to inhibit any RNase activity.

    Real-time Polymerase Chain Reaction:

    Article Title: Sterol Regulatory Element-binding Protein (SREBP) Cleavage Regulates Golgi-to-Endoplasmic Reticulum Recycling of SREBP Cleavage-activating Protein (SCAP) *
    Article Snippet: .. We obtained yeast extract, peptone, and agar from BD Biosciences; S1P inhibitor PF-429242 from Shanghai APIs Chemical Co.; proteasome inhibitor MG132 (C2211), lysosome inhibitor ammonium chloride (A9434), mevalonolactone (M4667, for sodium mevalonate preparation), puromycin dihydrochloride (P8833), oleic acid-albumin (O3008), doxycycline (D9891), crystal violet (C3886), soybean trypsin inhibitor (T9003), glass beads (G8772, for yeast cell lysis), trypsin (T8003), and lipoprotein-deficient serum (LPDS; S5394) from Sigma-Aldrich (catalogue numbers in parentheses); cell culture media DMEM (10-013), DMEM/F12 (10-092), and penicillin-streptomycin (30-002) from Corning Cellgro; FuGENE 6 and RNase-free DNase I (10104159001) from Roche Applied Science; random primer mix (S1330), M-MuLV reverse transcriptase (M0253L), murine RNase inhibitor (M0314L), oligo d(T)23 VN (S1327S), and endoglycosidase Hf (P0703) from New England Biolabs; GoTaq real-time PCR mix (A6002) from Promega; SCAP trafficking inhibitor fatostatin (341329) and compactin (mevastatin, 474705) from Millipore; and BioCoatTM collagen-coated culture dish (VWR 62405-617) from BD Biosciences. .. We obtained wild-type haploid S. pombe KGY425 from ATCC.

    Incubation:

    Article Title: Impaired spliceosomal UsnRNP assembly leads to Sm mRNA down-regulation and Sm protein degradation
    Article Snippet: The cells were then washed extensively using 1× PBS and lysed in lysis buffer (50 mM Hepes-NaOH, pH 7.5, 150 mM NaCl, 1% NP-40, 2.5 mM MgCl2 , 0.8 U/µl murine RNase inhibitor [M0314S; New England Biolabs, Inc.], and protease inhibitor cocktail) as described in pSILAC labeling. .. In parallel, the antibody against the protein of interest was incubated with protein G–Sepharose beads on a head-over-tail rotor for 1 h at RT and washed once with 1× PBS and twice with lysis buffer.

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
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    Article Title: Endothelial cell CD36 optimizes tissue fatty acid uptake
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    Article Title: Post-transcriptional 3´-UTR cleavage of mRNA transcripts generates thousands of stable uncapped autonomous RNA fragments
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    Article Title: The amino terminal extension of mammalian mitochondrial RNA polymerase ensures promoter specific transcription initiation
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    Article Title: 3′ end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character—RNA-Seq analyses
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    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
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    Activity Assay:

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein. .. Additionally, sialidase activity of in vitro expressed proteins was assessed by incubating 20 μl of PURExpress product with 4 μl of 100 μg/ml 4MU-α-Neu5Ac at 37 °C for 1 h and reading fluorescence at λex = 365 nm and λem = 445 nm in a SpectraMax microplate fluorometer.).

    Article Title: 3′ end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character—RNA-Seq analyses
    Article Snippet: .. RNase Inhibitor Murine (New England BioLabs) was added to each transcription mixture before and after the reaction, to inhibit any RNase activity. ..

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein. .. Additionally, sialidase activity of in vitro expressed proteins was assessed by incubating 20 μl of PURExpress product with 4 μl of 100 μg/ml 4MU-α-Neu5Ac at 37 °C for 1 h and reading fluorescence at λex = 365 nm and λem = 445 nm in a SpectraMax microplate fluorometer.

    Expressing:

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: Paragraph title: In vitro and in vivo sialidase expression ... For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein.

    Cell Fractionation:

    Article Title: A length-dependent evolutionarily conserved pathway controls nuclear export of circular RNAs
    Article Snippet: Cellular fractionation was performed as described previously ( ) with minor modifications. .. Briefly, cells were washed twice with serum-free medium (DL1 cells) or PBS (HeLa cells) and resuspended with slow pipetting in 1 mL of lysis buffer B (10 mM Tris-HCl at pH 8, 140 mM NaCl, 1.5 mM MgCl2 , 0.5% IGEPAL CA-630, 1 mM dithiothreitol, 80 U/mL RNase inhibitor [New England Biolabs, M0314L]).

    Gel Purification:

    Article Title: The amino terminal extension of mammalian mitochondrial RNA polymerase ensures promoter specific transcription initiation
    Article Snippet: The non-promoter template was produced by digestion of the LSP template with EcoRI followed by gel purification (Quiagen) of the vector fragment. .. All transcription reaction volumes were 25 μl and contained 25 mM Tris–HCl, pH 8.0, 10 mM MgCl2 , 64 mM NaCl, 100 μg/ml BSA, 1 mM DTT, 400 μM ATP, 150 μM GTP, 150 μM CTP 10 μM UTP, 0,02 μM α-32 P UTP (3000 Ci/mmol), 4 U RNase inhibitor Murine (New England Biolabs), 90 fmole of indicated template and proteins at indicated concentrations.

    Protease Inhibitor:

    Article Title: Ubiquitination-dependent control of sexual differentiation in fission yeast
    Article Snippet: .. Cell pellets were resuspended in 2 ml lysis buffer (6 mM Na2 HPO4 , 4 mM NaH2 PO4 , 150 mM NaC2 H3 O2 , 5 mM MgC2 H3 O2 , 0.25% NP-40, 2 mM EDTA, 1 mM EGTA, 5% glycerol, 1 mM AEBSF, 4 mM benzamidine, 2X Roche complete EDTA-free protease inhibitor cocktail and 160 U Murine RNase inhibitor (New England Biolabs, #M0314L)) to make ‘pop-corn’. ..

    Article Title: Impaired spliceosomal UsnRNP assembly leads to Sm mRNA down-regulation and Sm protein degradation
    Article Snippet: .. The cells were then washed extensively using 1× PBS and lysed in lysis buffer (50 mM Hepes-NaOH, pH 7.5, 150 mM NaCl, 1% NP-40, 2.5 mM MgCl2 , 0.8 U/µl murine RNase inhibitor [M0314S; New England Biolabs, Inc.], and protease inhibitor cocktail) as described in pSILAC labeling. .. Lysates were precleared using Sepharose–protein G beads (GE Healthcare) for 1 h at 4°C on a head-over-tail rotor.

    Cell Culture:

    Article Title: Sterol Regulatory Element-binding Protein (SREBP) Cleavage Regulates Golgi-to-Endoplasmic Reticulum Recycling of SREBP Cleavage-activating Protein (SCAP) *
    Article Snippet: .. We obtained yeast extract, peptone, and agar from BD Biosciences; S1P inhibitor PF-429242 from Shanghai APIs Chemical Co.; proteasome inhibitor MG132 (C2211), lysosome inhibitor ammonium chloride (A9434), mevalonolactone (M4667, for sodium mevalonate preparation), puromycin dihydrochloride (P8833), oleic acid-albumin (O3008), doxycycline (D9891), crystal violet (C3886), soybean trypsin inhibitor (T9003), glass beads (G8772, for yeast cell lysis), trypsin (T8003), and lipoprotein-deficient serum (LPDS; S5394) from Sigma-Aldrich (catalogue numbers in parentheses); cell culture media DMEM (10-013), DMEM/F12 (10-092), and penicillin-streptomycin (30-002) from Corning Cellgro; FuGENE 6 and RNase-free DNase I (10104159001) from Roche Applied Science; random primer mix (S1330), M-MuLV reverse transcriptase (M0253L), murine RNase inhibitor (M0314L), oligo d(T)23 VN (S1327S), and endoglycosidase Hf (P0703) from New England Biolabs; GoTaq real-time PCR mix (A6002) from Promega; SCAP trafficking inhibitor fatostatin (341329) and compactin (mevastatin, 474705) from Millipore; and BioCoatTM collagen-coated culture dish (VWR 62405-617) from BD Biosciences. .. We obtained wild-type haploid S. pombe KGY425 from ATCC.

    other:

    Article Title: Uridine Depletion and Chemical Modification Increase Cas9 mRNA Activity and Reduce Immunogenicity without HPLC Purification
    Article Snippet: Transcriptions were done in 1× transcription buffer (40 mM Tris, 10 mM dithiothreitol, 2 mM spermidine, 0.002% Triton X-100, and 27 mM magnesium acetate) using final concentrations of 8 U/μL T7 RNA polymerase (M0251L); 0.002 U/μL inorganic pyrophosphatase (M2403L); 1 U/μL murine RNase inhibitor (M0314L); 0.025 μg/μL standard or uridine-depleted transcription template; 5 mM CleanCap Cap 1 AG trimer; and 5 mM each of ATP, cytidine triphosphate (CTP) (or CTP analog), GTP, and uridine triphosphate (UTP) (or UTP analog), as indicated in .

    Sequencing:

    Article Title: 3′ end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character—RNA-Seq analyses
    Article Snippet: The reaction was incubated at 37°C for 4 h. Primer extension on synthetic 24-mer RNA (purchased from IDT, sequence is shown in ) was performed under the above ‘low yield’ condition, with 25 μM RNA replacing template DNA. .. RNase Inhibitor Murine (New England BioLabs) was added to each transcription mixture before and after the reaction, to inhibit any RNase activity.

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein. .. ORF12 was fused with a hexahistidine tag coding sequence at its 3′ end (for a C-terminal tag) and cloned into the pJS119K vector ( ).

    Binding Assay:

    Article Title: Post-transcriptional 3´-UTR cleavage of mRNA transcripts generates thousands of stable uncapped autonomous RNA fragments
    Article Snippet: .. After two washes with PBS, the anti-Flag-coated beads were added to the poly(A) selected RNA extract in 0.5 ml binding buffer (50 mM Tris, 150 mM NaCl, 0.5% Triton, and 1 µl Murine RNAse inhibitor (NEB-M0314S)) and incubated for 1.5 h in 4 °C. .. Precleared RNA samples were then subjected to 5′-CAP RNA immunoprecipitation using 5 µl (1:100 dilution) anti-Cap antibody (Merck anti-m3G-cap, m7G-cap antibody, clone H-20) and incubated O/N in 4 °C.

    In Vivo:

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: Paragraph title: In vitro and in vivo sialidase expression ... For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein.

    Fluorescence:

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein. .. Additionally, sialidase activity of in vitro expressed proteins was assessed by incubating 20 μl of PURExpress product with 4 μl of 100 μg/ml 4MU-α-Neu5Ac at 37 °C for 1 h and reading fluorescence at λex = 365 nm and λem = 445 nm in a SpectraMax microplate fluorometer.).

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein. .. Additionally, sialidase activity of in vitro expressed proteins was assessed by incubating 20 μl of PURExpress product with 4 μl of 100 μg/ml 4MU-α-Neu5Ac at 37 °C for 1 h and reading fluorescence at λex = 365 nm and λem = 445 nm in a SpectraMax microplate fluorometer.

    Isolation:

    Article Title: Endothelial cell CD36 optimizes tissue fatty acid uptake
    Article Snippet: CMs were pelleted and subjected to Percoll density gradient centrifugation (1,200 g for 30 min) for RNA and protein isolation. .. RBC in non-CMs were lysed and pellets resuspended in staining buffer (2% FBS/PBS, 2 mM EDTA, and 100 U/ml RNase inhibitor; catalog M0314L, New England BioLabs).

    Labeling:

    Article Title: Impaired spliceosomal UsnRNP assembly leads to Sm mRNA down-regulation and Sm protein degradation
    Article Snippet: .. The cells were then washed extensively using 1× PBS and lysed in lysis buffer (50 mM Hepes-NaOH, pH 7.5, 150 mM NaCl, 1% NP-40, 2.5 mM MgCl2 , 0.8 U/µl murine RNase inhibitor [M0314S; New England Biolabs, Inc.], and protease inhibitor cocktail) as described in pSILAC labeling. .. Lysates were precleared using Sepharose–protein G beads (GE Healthcare) for 1 h at 4°C on a head-over-tail rotor.

    Purification:

    Article Title: POLRMT regulates the switch between replication primer formation and gene expression of mammalian mtDNA
    Article Snippet: In vitro transcription assay Transcription reactions were in 25 μl total volume containing 25 mM tris-HCl (pH 8.0), 10 mM MgCl2 , 64 mM NaCl, BSA (100 μg/ml), 1 mM dithiothreitol (DTT), 400 μM ATP, 150 μM guanosine-5′-triphosphate (GTP), 150 μM cytidine-5′-triphosphate (CTP), 10 μM uridine 5′-triphosphate (UTP), 0.02 μM [α-32 P]UTP, and 4 U RNase Inhibitor Murine (New England Biolabs). .. The transcription template was added at 4 nM and consisted of a restriction cut (Hind III/Eco RI), purified (QIAquick PCR Purification Kit) human LSP/HSP plasmid, where a fragment consisting of positions 325 to 742 of human mtDNA was cloned between the Sma I and Hind III sites of pUC18 ( ).

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: The amplified product was purified using the Monarch PCR clean-up kit (New England Biolabs). .. For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein.

    Article Title: The amino terminal extension of mammalian mitochondrial RNA polymerase ensures promoter specific transcription initiation
    Article Snippet: All transcription reaction volumes were 25 μl and contained 25 mM Tris–HCl, pH 8.0, 10 mM MgCl2 , 64 mM NaCl, 100 μg/ml BSA, 1 mM DTT, 400 μM ATP, 150 μM GTP, 150 μM CTP 10 μM UTP, 0,02 μM α-32 P UTP (3000 Ci/mmol), 4 U RNase inhibitor Murine (New England Biolabs), 90 fmole of indicated template and proteins at indicated concentrations. .. The transcripts were purified with ethanol precipitation and the pellets were dissolved in 20 μl gel loading buffer (98% formamide, 10 mM EDTA, 0.025% xylene cyanol and 0.025% bromophenol blue) and heated at 95°C for 5 min.

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: The amplified product was purified using the Monarch PCR clean-up kit (New England Biolabs). .. For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein.

    Polymerase Chain Reaction:

    Article Title: POLRMT regulates the switch between replication primer formation and gene expression of mammalian mtDNA
    Article Snippet: In vitro transcription assay Transcription reactions were in 25 μl total volume containing 25 mM tris-HCl (pH 8.0), 10 mM MgCl2 , 64 mM NaCl, BSA (100 μg/ml), 1 mM dithiothreitol (DTT), 400 μM ATP, 150 μM guanosine-5′-triphosphate (GTP), 150 μM cytidine-5′-triphosphate (CTP), 10 μM uridine 5′-triphosphate (UTP), 0.02 μM [α-32 P]UTP, and 4 U RNase Inhibitor Murine (New England Biolabs). .. The transcription template was added at 4 nM and consisted of a restriction cut (Hind III/Eco RI), purified (QIAquick PCR Purification Kit) human LSP/HSP plasmid, where a fragment consisting of positions 325 to 742 of human mtDNA was cloned between the Sma I and Hind III sites of pUC18 ( ).

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: The amplified product was purified using the Monarch PCR clean-up kit (New England Biolabs). .. For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein.

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: The amplified product was purified using the Monarch PCR clean-up kit (New England Biolabs). .. For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein.

    FACS:

    Article Title: Endothelial cell CD36 optimizes tissue fatty acid uptake
    Article Snippet: Non-CMs in the supernatant were centrifuged at 500 g (10 min) and prepared for FACS. .. RBC in non-CMs were lysed and pellets resuspended in staining buffer (2% FBS/PBS, 2 mM EDTA, and 100 U/ml RNase inhibitor; catalog M0314L, New England BioLabs).

    Lysis:

    Article Title: Ubiquitination-dependent control of sexual differentiation in fission yeast
    Article Snippet: .. Cell pellets were resuspended in 2 ml lysis buffer (6 mM Na2 HPO4 , 4 mM NaH2 PO4 , 150 mM NaC2 H3 O2 , 5 mM MgC2 H3 O2 , 0.25% NP-40, 2 mM EDTA, 1 mM EGTA, 5% glycerol, 1 mM AEBSF, 4 mM benzamidine, 2X Roche complete EDTA-free protease inhibitor cocktail and 160 U Murine RNase inhibitor (New England Biolabs, #M0314L)) to make ‘pop-corn’. ..

    Article Title: Impaired spliceosomal UsnRNP assembly leads to Sm mRNA down-regulation and Sm protein degradation
    Article Snippet: .. The cells were then washed extensively using 1× PBS and lysed in lysis buffer (50 mM Hepes-NaOH, pH 7.5, 150 mM NaCl, 1% NP-40, 2.5 mM MgCl2 , 0.8 U/µl murine RNase inhibitor [M0314S; New England Biolabs, Inc.], and protease inhibitor cocktail) as described in pSILAC labeling. .. Lysates were precleared using Sepharose–protein G beads (GE Healthcare) for 1 h at 4°C on a head-over-tail rotor.

    Article Title: Sterol Regulatory Element-binding Protein (SREBP) Cleavage Regulates Golgi-to-Endoplasmic Reticulum Recycling of SREBP Cleavage-activating Protein (SCAP) *
    Article Snippet: .. We obtained yeast extract, peptone, and agar from BD Biosciences; S1P inhibitor PF-429242 from Shanghai APIs Chemical Co.; proteasome inhibitor MG132 (C2211), lysosome inhibitor ammonium chloride (A9434), mevalonolactone (M4667, for sodium mevalonate preparation), puromycin dihydrochloride (P8833), oleic acid-albumin (O3008), doxycycline (D9891), crystal violet (C3886), soybean trypsin inhibitor (T9003), glass beads (G8772, for yeast cell lysis), trypsin (T8003), and lipoprotein-deficient serum (LPDS; S5394) from Sigma-Aldrich (catalogue numbers in parentheses); cell culture media DMEM (10-013), DMEM/F12 (10-092), and penicillin-streptomycin (30-002) from Corning Cellgro; FuGENE 6 and RNase-free DNase I (10104159001) from Roche Applied Science; random primer mix (S1330), M-MuLV reverse transcriptase (M0253L), murine RNase inhibitor (M0314L), oligo d(T)23 VN (S1327S), and endoglycosidase Hf (P0703) from New England Biolabs; GoTaq real-time PCR mix (A6002) from Promega; SCAP trafficking inhibitor fatostatin (341329) and compactin (mevastatin, 474705) from Millipore; and BioCoatTM collagen-coated culture dish (VWR 62405-617) from BD Biosciences. .. We obtained wild-type haploid S. pombe KGY425 from ATCC.

    Article Title: A length-dependent evolutionarily conserved pathway controls nuclear export of circular RNAs
    Article Snippet: .. Briefly, cells were washed twice with serum-free medium (DL1 cells) or PBS (HeLa cells) and resuspended with slow pipetting in 1 mL of lysis buffer B (10 mM Tris-HCl at pH 8, 140 mM NaCl, 1.5 mM MgCl2 , 0.5% IGEPAL CA-630, 1 mM dithiothreitol, 80 U/mL RNase inhibitor [New England Biolabs, M0314L]). ..

    Plasmid Preparation:

    Article Title: POLRMT regulates the switch between replication primer formation and gene expression of mammalian mtDNA
    Article Snippet: In vitro transcription assay Transcription reactions were in 25 μl total volume containing 25 mM tris-HCl (pH 8.0), 10 mM MgCl2 , 64 mM NaCl, BSA (100 μg/ml), 1 mM dithiothreitol (DTT), 400 μM ATP, 150 μM guanosine-5′-triphosphate (GTP), 150 μM cytidine-5′-triphosphate (CTP), 10 μM uridine 5′-triphosphate (UTP), 0.02 μM [α-32 P]UTP, and 4 U RNase Inhibitor Murine (New England Biolabs). .. The transcription template was added at 4 nM and consisted of a restriction cut (Hind III/Eco RI), purified (QIAquick PCR Purification Kit) human LSP/HSP plasmid, where a fragment consisting of positions 325 to 742 of human mtDNA was cloned between the Sma I and Hind III sites of pUC18 ( ).

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein. .. Linearization of the vector was performed by PCR with Q5 hot start high-fidelity 2× Master Mix (New England Biolabs) for 25 cycles (98 °C for 10 s, 60 °C for 30 s, and 72 °C for 2 min).

    Article Title: Mitochondrial transcription termination factor 1 directs polar replication fork pausing
    Article Snippet: .. The transcription reaction volumes were 25 μl and contained 25 mM Tris-HCl pH 8.0, 10 mM MgCl2 , 40 mM NaCl, 100 μg/mL BSA, 10 mM DTT, 400 μM ATP, 150 μM GTP, 150 μM CTP 10 μM UTP, 0,02 μM α-32 P UTP (3000 Ci/mmol), 4 U RNase inhibitor Murine (New England Biolabs), 4 nM of indicated plasmid template. ..

    Article Title: The amino terminal extension of mammalian mitochondrial RNA polymerase ensures promoter specific transcription initiation
    Article Snippet: The non-promoter template was produced by digestion of the LSP template with EcoRI followed by gel purification (Quiagen) of the vector fragment. .. All transcription reaction volumes were 25 μl and contained 25 mM Tris–HCl, pH 8.0, 10 mM MgCl2 , 64 mM NaCl, 100 μg/ml BSA, 1 mM DTT, 400 μM ATP, 150 μM GTP, 150 μM CTP 10 μM UTP, 0,02 μM α-32 P UTP (3000 Ci/mmol), 4 U RNase inhibitor Murine (New England Biolabs), 90 fmole of indicated template and proteins at indicated concentrations.

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein. .. ORF12 was fused with a hexahistidine tag coding sequence at its 3′ end (for a C-terminal tag) and cloned into the pJS119K vector ( ).

    In Vitro:

    Article Title: POLRMT regulates the switch between replication primer formation and gene expression of mammalian mtDNA
    Article Snippet: .. In vitro transcription assay Transcription reactions were in 25 μl total volume containing 25 mM tris-HCl (pH 8.0), 10 mM MgCl2 , 64 mM NaCl, BSA (100 μg/ml), 1 mM dithiothreitol (DTT), 400 μM ATP, 150 μM guanosine-5′-triphosphate (GTP), 150 μM cytidine-5′-triphosphate (CTP), 10 μM uridine 5′-triphosphate (UTP), 0.02 μM [α-32 P]UTP, and 4 U RNase Inhibitor Murine (New England Biolabs). .. The transcription template was added at 4 nM and consisted of a restriction cut (Hind III/Eco RI), purified (QIAquick PCR Purification Kit) human LSP/HSP plasmid, where a fragment consisting of positions 325 to 742 of human mtDNA was cloned between the Sma I and Hind III sites of pUC18 ( ).

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: .. For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein. .. Expression was verified by separating 2.5 μl of the reaction on a Novex 10–20% Tris–glycine gel (Thermo Fisher Scientific).

    Article Title: Mitochondrial transcription termination factor 1 directs polar replication fork pausing
    Article Snippet: Paragraph title: In vitro transcription experiments ... The transcription reaction volumes were 25 μl and contained 25 mM Tris-HCl pH 8.0, 10 mM MgCl2 , 40 mM NaCl, 100 μg/mL BSA, 10 mM DTT, 400 μM ATP, 150 μM GTP, 150 μM CTP 10 μM UTP, 0,02 μM α-32 P UTP (3000 Ci/mmol), 4 U RNase inhibitor Murine (New England Biolabs), 4 nM of indicated plasmid template.

    Article Title: The amino terminal extension of mammalian mitochondrial RNA polymerase ensures promoter specific transcription initiation
    Article Snippet: Paragraph title: In vitro transcription ... All transcription reaction volumes were 25 μl and contained 25 mM Tris–HCl, pH 8.0, 10 mM MgCl2 , 64 mM NaCl, 100 μg/ml BSA, 1 mM DTT, 400 μM ATP, 150 μM GTP, 150 μM CTP 10 μM UTP, 0,02 μM α-32 P UTP (3000 Ci/mmol), 4 U RNase inhibitor Murine (New England Biolabs), 90 fmole of indicated template and proteins at indicated concentrations.

    Article Title: 3′ end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character—RNA-Seq analyses
    Article Snippet: Paragraph title: In vitro transcription by T7 RNA polymerase ... RNase Inhibitor Murine (New England BioLabs) was added to each transcription mixture before and after the reaction, to inhibit any RNase activity.

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: .. For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein. .. Expression was verified by separating 2.5 μl of the reaction on a Novex 10–20% Tris–glycine gel (Thermo Fisher Scientific).

    Ethanol Precipitation:

    Article Title: The amino terminal extension of mammalian mitochondrial RNA polymerase ensures promoter specific transcription initiation
    Article Snippet: All transcription reaction volumes were 25 μl and contained 25 mM Tris–HCl, pH 8.0, 10 mM MgCl2 , 64 mM NaCl, 100 μg/ml BSA, 1 mM DTT, 400 μM ATP, 150 μM GTP, 150 μM CTP 10 μM UTP, 0,02 μM α-32 P UTP (3000 Ci/mmol), 4 U RNase inhibitor Murine (New England Biolabs), 90 fmole of indicated template and proteins at indicated concentrations. .. The transcripts were purified with ethanol precipitation and the pellets were dissolved in 20 μl gel loading buffer (98% formamide, 10 mM EDTA, 0.025% xylene cyanol and 0.025% bromophenol blue) and heated at 95°C for 5 min.

    Produced:

    Article Title: The amino terminal extension of mammalian mitochondrial RNA polymerase ensures promoter specific transcription initiation
    Article Snippet: The non-promoter template was produced by digestion of the LSP template with EcoRI followed by gel purification (Quiagen) of the vector fragment. .. All transcription reaction volumes were 25 μl and contained 25 mM Tris–HCl, pH 8.0, 10 mM MgCl2 , 64 mM NaCl, 100 μg/ml BSA, 1 mM DTT, 400 μM ATP, 150 μM GTP, 150 μM CTP 10 μM UTP, 0,02 μM α-32 P UTP (3000 Ci/mmol), 4 U RNase inhibitor Murine (New England Biolabs), 90 fmole of indicated template and proteins at indicated concentrations.

    Immunoprecipitation:

    Article Title: Impaired spliceosomal UsnRNP assembly leads to Sm mRNA down-regulation and Sm protein degradation
    Article Snippet: Paragraph title: 35 S metabolic labeling, immunoprecipitation, and autoradiography ... The cells were then washed extensively using 1× PBS and lysed in lysis buffer (50 mM Hepes-NaOH, pH 7.5, 150 mM NaCl, 1% NP-40, 2.5 mM MgCl2 , 0.8 U/µl murine RNase inhibitor [M0314S; New England Biolabs, Inc.], and protease inhibitor cocktail) as described in pSILAC labeling.

    Article Title: Post-transcriptional 3´-UTR cleavage of mRNA transcripts generates thousands of stable uncapped autonomous RNA fragments
    Article Snippet: Paragraph title: 5′- CAP RNA immunoprecipitation ... After two washes with PBS, the anti-Flag-coated beads were added to the poly(A) selected RNA extract in 0.5 ml binding buffer (50 mM Tris, 150 mM NaCl, 0.5% Triton, and 1 µl Murine RNAse inhibitor (NEB-M0314S)) and incubated for 1.5 h in 4 °C.

    Fractionation:

    Article Title: Endothelial cell CD36 optimizes tissue fatty acid uptake
    Article Snippet: Paragraph title: Heart suborgan fractionation. ... RBC in non-CMs were lysed and pellets resuspended in staining buffer (2% FBS/PBS, 2 mM EDTA, and 100 U/ml RNase inhibitor; catalog M0314L, New England BioLabs).

    Article Title: A length-dependent evolutionarily conserved pathway controls nuclear export of circular RNAs
    Article Snippet: Paragraph title: Nuclear and cytoplasmic fractionation ... Briefly, cells were washed twice with serum-free medium (DL1 cells) or PBS (HeLa cells) and resuspended with slow pipetting in 1 mL of lysis buffer B (10 mM Tris-HCl at pH 8, 140 mM NaCl, 1.5 mM MgCl2 , 0.5% IGEPAL CA-630, 1 mM dithiothreitol, 80 U/mL RNase inhibitor [New England Biolabs, M0314L]).

    Staining:

    Article Title: Endothelial cell CD36 optimizes tissue fatty acid uptake
    Article Snippet: .. RBC in non-CMs were lysed and pellets resuspended in staining buffer (2% FBS/PBS, 2 mM EDTA, and 100 U/ml RNase inhibitor; catalog M0314L, New England BioLabs). .. Incubation with calcein AM (100 nM, room temperature, 30 min) and then with CD45 and CD31 antibodies (catalogs 103114 and 102508, BioLegend) on ice (30 min) was followed by 7-AAD (5 μg/ml) for an additional 10 minutes before the end of the incubation.

    Gradient Centrifugation:

    Article Title: Endothelial cell CD36 optimizes tissue fatty acid uptake
    Article Snippet: CMs were pelleted and subjected to Percoll density gradient centrifugation (1,200 g for 30 min) for RNA and protein isolation. .. RBC in non-CMs were lysed and pellets resuspended in staining buffer (2% FBS/PBS, 2 mM EDTA, and 100 U/ml RNase inhibitor; catalog M0314L, New England BioLabs).

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    New England Biolabs murine rnase inhibitor
    Biochemical characterization of LwaCas13a <t>RNA</t> cleavage activity a, LwaCas13a has more active <t>RNAse</t> activity than LshCas13a. b, Gel electrophoresis of ssRNA1 after incubation with LwaCas13a and with and without crRNA 1 for varying amounts of times. c, Gel electrophoresis of ssRNA1 after incubation with varying amounts of LwaCas13a-crRNA complex. d, Sequence and structure of ssRNA 4 and ssRNA 5. crRNA spacer sequence is highlighted in blue. e, Gel electrophoresis of ssRNA 4 and ssRNA 5 after incubation with LwaCas13a and crRNA 1. f, Sequence and structure of ssRNA 4 with sites of poly-x modifications highlighted in red. crRNA spacer sequence is highlighted in blue. g, Gel electrophoresis of ssRNA 4 with each of 4 possible poly-x modifications incubated with LwaCas13a and crRNA 1. h, LwaCas13a can process pre-crRNA from the L. wadei CRISPR-Cas locus. i, Cleavage efficiency of ssRNA 1 for crRNA spacer truncations after incubation with LwaCas13a.
    Murine Rnase Inhibitor, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biochemical characterization of LwaCas13a RNA cleavage activity a, LwaCas13a has more active RNAse activity than LshCas13a. b, Gel electrophoresis of ssRNA1 after incubation with LwaCas13a and with and without crRNA 1 for varying amounts of times. c, Gel electrophoresis of ssRNA1 after incubation with varying amounts of LwaCas13a-crRNA complex. d, Sequence and structure of ssRNA 4 and ssRNA 5. crRNA spacer sequence is highlighted in blue. e, Gel electrophoresis of ssRNA 4 and ssRNA 5 after incubation with LwaCas13a and crRNA 1. f, Sequence and structure of ssRNA 4 with sites of poly-x modifications highlighted in red. crRNA spacer sequence is highlighted in blue. g, Gel electrophoresis of ssRNA 4 with each of 4 possible poly-x modifications incubated with LwaCas13a and crRNA 1. h, LwaCas13a can process pre-crRNA from the L. wadei CRISPR-Cas locus. i, Cleavage efficiency of ssRNA 1 for crRNA spacer truncations after incubation with LwaCas13a.

    Journal: Nature

    Article Title: RNA targeting with CRISPR-Cas13a

    doi: 10.1038/nature24049

    Figure Lengend Snippet: Biochemical characterization of LwaCas13a RNA cleavage activity a, LwaCas13a has more active RNAse activity than LshCas13a. b, Gel electrophoresis of ssRNA1 after incubation with LwaCas13a and with and without crRNA 1 for varying amounts of times. c, Gel electrophoresis of ssRNA1 after incubation with varying amounts of LwaCas13a-crRNA complex. d, Sequence and structure of ssRNA 4 and ssRNA 5. crRNA spacer sequence is highlighted in blue. e, Gel electrophoresis of ssRNA 4 and ssRNA 5 after incubation with LwaCas13a and crRNA 1. f, Sequence and structure of ssRNA 4 with sites of poly-x modifications highlighted in red. crRNA spacer sequence is highlighted in blue. g, Gel electrophoresis of ssRNA 4 with each of 4 possible poly-x modifications incubated with LwaCas13a and crRNA 1. h, LwaCas13a can process pre-crRNA from the L. wadei CRISPR-Cas locus. i, Cleavage efficiency of ssRNA 1 for crRNA spacer truncations after incubation with LwaCas13a.

    Article Snippet: Briefly, reactions consisted of 45 nM purified LwaCas13a, 22.5 nM crRNA, 125 nM quenched fluorescent RNA reporter (RNAse Alert v2, Thermo Scientific), 2 μL murine RNase inhibitor (New England Biolabs), 100 ng of background total human RNA (purified from HEK293FT culture), and varying amounts of input nucleic acid target, unless otherwise indicated, in nuclease assay buffer (40 mM Tris-HCl, 60 mM NaCl, 6 mM MgCl2, pH 7.3).

    Techniques: Activity Assay, Nucleic Acid Electrophoresis, Incubation, Sequencing, CRISPR

    Effect of the growth parameters on RiCF. (A) Schematics of a hypothetical scenario when RNA inhibits NAPs that could potentially cleave DNA. During lysis, quick RNA degradation removes the inhibition resulting in breakage of chromosomes. (B) Growth phase dependence of RiCF. AB1157 was grown at 37°C with periodic OD measurements, and samples for plugs were withdrawn at various times. The cells were made into plugs using lysis agarose and RNase (50 μg/plug) and the plugs were lysed and electrophoresed under standard conditions. Data points are means of at least three independent assays ± SEM. (C) Effect of translation and transcription inhibition on RiCF. Cells were grown till OD 0.5–0.6, split into three parts and chloramphenicol (40 μg/ml) or rifampicin (150 μg/ml) were added to two samples. All sample were shaken for another 2–3 hours at 37°C before making plugs as described in (B). Data points are means of four independent assays ± SEM. (D) Growth in minimal medium reduces RiCF. Cells were grown in LB or MOPS till the OD reached 0.6 and made into plugs using standard conditions. The values presented are means of six independent assays ± SEM. (E) Effect of growth temperature on RNase-induced chromosomal fragmentation. Cultures of AB1157 were grown at 20°C, 30°C, 37°C, 42°C or 45°C to same cell densities (A 600 = 0.6), and plugs were made in lysis agarose with RNAse A (50 μg/plug), as described in (A). Data are means of three to six independent measurements ± SEM.

    Journal: PLoS ONE

    Article Title: Degradation of RNA during lysis of Escherichia coli cells in agarose plugs breaks the chromosome

    doi: 10.1371/journal.pone.0190177

    Figure Lengend Snippet: Effect of the growth parameters on RiCF. (A) Schematics of a hypothetical scenario when RNA inhibits NAPs that could potentially cleave DNA. During lysis, quick RNA degradation removes the inhibition resulting in breakage of chromosomes. (B) Growth phase dependence of RiCF. AB1157 was grown at 37°C with periodic OD measurements, and samples for plugs were withdrawn at various times. The cells were made into plugs using lysis agarose and RNase (50 μg/plug) and the plugs were lysed and electrophoresed under standard conditions. Data points are means of at least three independent assays ± SEM. (C) Effect of translation and transcription inhibition on RiCF. Cells were grown till OD 0.5–0.6, split into three parts and chloramphenicol (40 μg/ml) or rifampicin (150 μg/ml) were added to two samples. All sample were shaken for another 2–3 hours at 37°C before making plugs as described in (B). Data points are means of four independent assays ± SEM. (D) Growth in minimal medium reduces RiCF. Cells were grown in LB or MOPS till the OD reached 0.6 and made into plugs using standard conditions. The values presented are means of six independent assays ± SEM. (E) Effect of growth temperature on RNase-induced chromosomal fragmentation. Cultures of AB1157 were grown at 20°C, 30°C, 37°C, 42°C or 45°C to same cell densities (A 600 = 0.6), and plugs were made in lysis agarose with RNAse A (50 μg/plug), as described in (A). Data are means of three to six independent measurements ± SEM.

    Article Snippet: XRN-I, RNase If , Exonuclease T (Exo T), EcoRI and RNase A inhibitor were all from New England Biolabs.

    Techniques: Lysis, Inhibition

    RNA degradation causes chromosomal fragmentation. (A)  Schematics of a hypothetical scenario when RNA makes the central core of nucleoids, and its degradation results in collapse of the nucleoid structure, causing chromosomal fragmentation.  (B)  Radiogram of a pulsed field gel showing chromosomal fragmentation in AB1157 when cells were embedded in agarose plugs in the presence and absence of proteinase K (25 μg/plug) and/or RNase (50 μg/plug) and lysed overnight at 62°C.  (C)  Radiogram showing DNase I sensitivity of the signal entering the gel. Plugs were lysed at 62°C, washed extensively to remove traces of lysis buffer and then treated with DNase I at 37°C before PFGE.  (D)  A representative gel showing that RNA degradation by different enzymes causes chromosomal fragmentation. Plugs were made in the absence of proteinase K in 1x restriction enzyme buffer (NEBuffer 3 for RNase A, XRN-1 and RNAse I f  and NEBuffer 4 for Exo T). The concentrations of the enzymes used were, RNase, 50 μg/plug; XRN-1, 5 U/plug; RNAse I f , 100 U/plug and Exo T, 20 U/plug.  (E)  Quantification of the chromosomal fragmentation when plugs were made in the presence of various RNA degrading enzymes. The values presented are means of four independent assays ± SEM. CZ, compression zone.

    Journal: PLoS ONE

    Article Title: Degradation of RNA during lysis of Escherichia coli cells in agarose plugs breaks the chromosome

    doi: 10.1371/journal.pone.0190177

    Figure Lengend Snippet: RNA degradation causes chromosomal fragmentation. (A) Schematics of a hypothetical scenario when RNA makes the central core of nucleoids, and its degradation results in collapse of the nucleoid structure, causing chromosomal fragmentation. (B) Radiogram of a pulsed field gel showing chromosomal fragmentation in AB1157 when cells were embedded in agarose plugs in the presence and absence of proteinase K (25 μg/plug) and/or RNase (50 μg/plug) and lysed overnight at 62°C. (C) Radiogram showing DNase I sensitivity of the signal entering the gel. Plugs were lysed at 62°C, washed extensively to remove traces of lysis buffer and then treated with DNase I at 37°C before PFGE. (D) A representative gel showing that RNA degradation by different enzymes causes chromosomal fragmentation. Plugs were made in the absence of proteinase K in 1x restriction enzyme buffer (NEBuffer 3 for RNase A, XRN-1 and RNAse I f and NEBuffer 4 for Exo T). The concentrations of the enzymes used were, RNase, 50 μg/plug; XRN-1, 5 U/plug; RNAse I f , 100 U/plug and Exo T, 20 U/plug. (E) Quantification of the chromosomal fragmentation when plugs were made in the presence of various RNA degrading enzymes. The values presented are means of four independent assays ± SEM. CZ, compression zone.

    Article Snippet: XRN-I, RNase If , Exonuclease T (Exo T), EcoRI and RNase A inhibitor were all from New England Biolabs.

    Techniques: Pulsed-Field Gel, Lysis

    eIF3d cap-binding activity is required for efficient 48S initiation complex formation on specific mRNAs a , Phosphorimage of SDS gel resolving RNase-protected 32 P-cap-labeled c-Jun 5′ UTR RNA crosslinked to eIF3 in the presence of competitor ligands. b , Electrostatic surface view of the eIF3d cap-binding domain colored by charge, with a zoomed view of single stranded RNA (ssRNA) and cap analog modeled according to their positions bound to DXO 15 . Positive charge is colored blue and negative charge is in red, and the RNA gate is removed for clarity. c , Phosphorimage of SDS gel resolving RNase-protected 32 P-cap-labeled c-Jun 5′ UTR RNA crosslinked to wild type or helix α5 or helix α11-mutant eIF3. eIF3d-helix α5 mutant (D249Q/V262I/Y263A), helix α11 mutant (T317E/N320E/H321A). WT, wild type. d , Incorporation of c-Jun and ACTB mRNA into initiation complexes by wild type, helix α5, or helix α11-mutant eIF3d as measured by quantitative RT-PCR. mRNA-ribosome association is expressed as the ratio between the quantity of mRNA transcripts to 18S rRNA and normalized to the wild type sample. The results are representative of three independent experiments and given as the mean ± s.d. from a representative quantitative RT-PCR experiment performed in duplicate.

    Journal: Nature

    Article Title: eIF3d is an mRNA cap-binding protein required for specialized translation initiation

    doi: 10.1038/nature18954

    Figure Lengend Snippet: eIF3d cap-binding activity is required for efficient 48S initiation complex formation on specific mRNAs a , Phosphorimage of SDS gel resolving RNase-protected 32 P-cap-labeled c-Jun 5′ UTR RNA crosslinked to eIF3 in the presence of competitor ligands. b , Electrostatic surface view of the eIF3d cap-binding domain colored by charge, with a zoomed view of single stranded RNA (ssRNA) and cap analog modeled according to their positions bound to DXO 15 . Positive charge is colored blue and negative charge is in red, and the RNA gate is removed for clarity. c , Phosphorimage of SDS gel resolving RNase-protected 32 P-cap-labeled c-Jun 5′ UTR RNA crosslinked to wild type or helix α5 or helix α11-mutant eIF3. eIF3d-helix α5 mutant (D249Q/V262I/Y263A), helix α11 mutant (T317E/N320E/H321A). WT, wild type. d , Incorporation of c-Jun and ACTB mRNA into initiation complexes by wild type, helix α5, or helix α11-mutant eIF3d as measured by quantitative RT-PCR. mRNA-ribosome association is expressed as the ratio between the quantity of mRNA transcripts to 18S rRNA and normalized to the wild type sample. The results are representative of three independent experiments and given as the mean ± s.d. from a representative quantitative RT-PCR experiment performed in duplicate.

    Article Snippet: Each translation reaction contained 50% in vitro translation lysate and buffer to make the final reaction with 0.84 mM ATP, 0.21 mM GTP, 21 mM creatine phosphate (Roche), 45 U ml-1 creatine phosphokinase (Roche), 10 mM HEPES-KOH pH 7.6, 2 mM DTT, 8 mM amino acids (Promega), 255 mM spermidine, 1 U ml-1 murine RNase inhibitor (NEB), and mRNA-specific concentrations of Mg(OAc)2 and KOAc.

    Techniques: Binding Assay, Activity Assay, SDS-Gel, Labeling, Mutagenesis, Quantitative RT-PCR

    eIF4E recognizes the 5′ end of the c-Jun mRNA less efficiently than ACTB mRNA a , Coomassie blue stained SDS gel of recombinant human eIF4E expressed in E. coli. b , Phosphorimage of SDS gel resolving RNase-protected 32 P-cap-labeled ACTB or c-Jun 5′ UTR RNA crosslinked to eIF4E. The result is representative of three independent experiments. For gel source data, see Supplementary Figure 1 .

    Journal: Nature

    Article Title: eIF3d is an mRNA cap-binding protein required for specialized translation initiation

    doi: 10.1038/nature18954

    Figure Lengend Snippet: eIF4E recognizes the 5′ end of the c-Jun mRNA less efficiently than ACTB mRNA a , Coomassie blue stained SDS gel of recombinant human eIF4E expressed in E. coli. b , Phosphorimage of SDS gel resolving RNase-protected 32 P-cap-labeled ACTB or c-Jun 5′ UTR RNA crosslinked to eIF4E. The result is representative of three independent experiments. For gel source data, see Supplementary Figure 1 .

    Article Snippet: Each translation reaction contained 50% in vitro translation lysate and buffer to make the final reaction with 0.84 mM ATP, 0.21 mM GTP, 21 mM creatine phosphate (Roche), 45 U ml-1 creatine phosphokinase (Roche), 10 mM HEPES-KOH pH 7.6, 2 mM DTT, 8 mM amino acids (Promega), 255 mM spermidine, 1 U ml-1 murine RNase inhibitor (NEB), and mRNA-specific concentrations of Mg(OAc)2 and KOAc.

    Techniques: Staining, SDS-Gel, Recombinant, Labeling

    5' end recognition of c-Jun mRNA is eIF4F-independent a , Distribution of c-Jun or ACTB mRNA-containing initiation complexes in programmed 293T cell in vitro translation extracts. The mRNA abundance (black line) is expressed as the fraction of total recovered transcripts. The results are given as the mean ± standard deviation (s.d.) of a representative quantitative RT-PCR experiment performed in duplicate. The polysome profile (gray line) is plotted as relative absorbance at 254 nm versus elution fractions. b , Western blot analysis of initiation factors in 48S translation complexes formed on c-Jun and ACTB mRNAs. 293T, total protein from 293T in vitro translation extracts. For gel source data, see Supplementary Figure 1 . c , Phosphorimage of SDS gel resolving RNase-protected 32 P-internal or 32 P-cap-labeled c-Jun 5' UTR RNA crosslinked to eIF3 subunits. Recombinant eIF3a migrates at ~100 kDa due to a C-terminal truncation 26 . The results of a - c are representative of three independent experiments.

    Journal: Nature

    Article Title: eIF3d is an mRNA cap-binding protein required for specialized translation initiation

    doi: 10.1038/nature18954

    Figure Lengend Snippet: 5' end recognition of c-Jun mRNA is eIF4F-independent a , Distribution of c-Jun or ACTB mRNA-containing initiation complexes in programmed 293T cell in vitro translation extracts. The mRNA abundance (black line) is expressed as the fraction of total recovered transcripts. The results are given as the mean ± standard deviation (s.d.) of a representative quantitative RT-PCR experiment performed in duplicate. The polysome profile (gray line) is plotted as relative absorbance at 254 nm versus elution fractions. b , Western blot analysis of initiation factors in 48S translation complexes formed on c-Jun and ACTB mRNAs. 293T, total protein from 293T in vitro translation extracts. For gel source data, see Supplementary Figure 1 . c , Phosphorimage of SDS gel resolving RNase-protected 32 P-internal or 32 P-cap-labeled c-Jun 5' UTR RNA crosslinked to eIF3 subunits. Recombinant eIF3a migrates at ~100 kDa due to a C-terminal truncation 26 . The results of a - c are representative of three independent experiments.

    Article Snippet: Each translation reaction contained 50% in vitro translation lysate and buffer to make the final reaction with 0.84 mM ATP, 0.21 mM GTP, 21 mM creatine phosphate (Roche), 45 U ml-1 creatine phosphokinase (Roche), 10 mM HEPES-KOH pH 7.6, 2 mM DTT, 8 mM amino acids (Promega), 255 mM spermidine, 1 U ml-1 murine RNase inhibitor (NEB), and mRNA-specific concentrations of Mg(OAc)2 and KOAc.

    Techniques: In Vitro, Standard Deviation, Quantitative RT-PCR, Western Blot, SDS-Gel, Labeling, Recombinant

    CBP interacts with RNA in vivo A) Native RNA-IP of CBP. Top, RNA immunprecipitated with CBP. Bottom, CBP western blot. B) PAR-CLIP protocol. 4-Thiouridine (4-SU). C) CBP PAR-CLIP required 4-SU: top, autoradiography; bottom, CBP western blot.. D) Quantification of CBP PAR-CLIP. Error bars represent mean +/− s.e.m; n=4. E) CBP PAR-CLIP signal was sensitive to RNAse. 1× RNAse cocktail contained: RNAse A (0.01mU/ul) + RNase T1 (0.4mU/ul). F) Quantification of RNase titration. Error bars represent mean +/− s.e.m; n=4; P -values from two-tailed Student’s t-test: *P

    Journal: Cell

    Article Title: RNA binding to CBP stimulates histone acetylation and transcription

    doi: 10.1016/j.cell.2016.12.020

    Figure Lengend Snippet: CBP interacts with RNA in vivo A) Native RNA-IP of CBP. Top, RNA immunprecipitated with CBP. Bottom, CBP western blot. B) PAR-CLIP protocol. 4-Thiouridine (4-SU). C) CBP PAR-CLIP required 4-SU: top, autoradiography; bottom, CBP western blot.. D) Quantification of CBP PAR-CLIP. Error bars represent mean +/− s.e.m; n=4. E) CBP PAR-CLIP signal was sensitive to RNAse. 1× RNAse cocktail contained: RNAse A (0.01mU/ul) + RNase T1 (0.4mU/ul). F) Quantification of RNase titration. Error bars represent mean +/− s.e.m; n=4; P -values from two-tailed Student’s t-test: *P

    Article Snippet: Reactions contained 1× HAT assay buffer (50mM Tris-HCl pH 7.5 (RT), 5% glycerol, 0.1mM EDTA, 50mM KCl), 1mM DTT, 10mM Na-Butyrate, 1× Complete EDTA protease inhibitor cocktail (Roche), 0.4U/ul murine RNAse inhibitor (NEB), 0.1mg/ml BSA (NEB), 80nM H31–21 peptide (Anaspec) and the required dilution of RNA probe.

    Techniques: In Vivo, Western Blot, Cross-linking Immunoprecipitation, Autoradiography, Titration, Two Tailed Test