5 ethynyl uridine  (Jena Bioscience)


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    Name:
    5 Ethynyl uridine
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    CLK-N002-10
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    Structured Review

    Jena Bioscience 5 ethynyl uridine
    More RNA polymerases I‐III means higher germination ability and GFP inducibility Protocol for (B). See 5‐Ethynyl Uridine (5‐EU; denoted U*) that incorporates into freshly made RNAs. We fix the spores afterwards and let fluorophore (Alexa 488) enter the spores and bind U* (see Materials and Methods). "Total RNA level" of a spore bag is th e total fluorescence from all the 5‐EU labelled RNAs. Microscope images show a spore bag (from strain “TS3”) after either incorporating 5‐EU as described in (C) (top two images) or, as a control, following the protocol in (C) but without the 5‐EU (bottom two images) (also see Appendix Fig S23 A). Scale bar: 2 μm. Top histogram: total RNA level per spore bag with 5‐EU (i.e. Alexa 488 fluorescence; n = 103 spore bags). Bottom histogram: fluorescence per spore bag in control population (i.e. without 5‐EU; n = 95 spore bags). Also see Appendix Fig S22 A. Each dot is from a single spore bag ("TS3" strain) with GFP‐inducing circuit (Fig 3A ). For each spore bag, we measured its GFP protein level ("GFP inducibility") and total RNA level (5‐EU fluorescence) after incubating the spores for 24 h in PBS with both 100‐μg/ml doxycycline and 1 mM of 5‐EU (see (C)). n = 245 spore bags. Alexa 594 fluorophore attached to 5‐EU (see Materials and Methods). Green line: linear regression with R = 0.24 and Pearson P ‐value = 0.00018. See "Protocol for Fig 4F " in Materials and Methods. GFP inducibility per spore bag (each dot, "TS8" strain) measured as in (E) but now with live time‐lapse without the 5‐EU. "RNAP II level" is the mCherry fluorescence per spore bag due to the mCherry protein fused to Rpb3, a subunit of RNA polymerase II. n = 182 spore bags; Red line: linear regression with R = 0.64 and Pearson P ‐value = 3.02 × 10 −22 . Scale bar = 2 μm. Same protocol as in (F) but with "TS9" spores. "RNAP I III level" is the mCherry fluorescence per spore bag due to the mCherry protein fused to Rpc40, a subunit of both RNAP I and RNAP III. n = 185 spore bags; Red line: linear regression with R = 0.63 and Pearson P ‐value = 6.6 × 10 −22 . Scale bar = 2 μm. Same protocol as in (F) but with "TT14" spores fixed after 24 h of incubation in PBS with 100‐μg/ml doxycycline. 18s rRNA level is from CAL Fluor Red 610 fluorescence emitted by single‐molecule FISH probes bound to 18S rRNAs (see Materials and Methods). n = 213 spore bags; Red line: linear regression with R = 0.19 and Pearson P ‐value = 0.005. Scale bar = 2 μm. Bottom: RNAP II levels of spore bags ("TS8" strain) in a population, measured as in (F). Top: As a function of the RNAP II level (binned in the histogram), percentage of spore bags that germinated after receiving a 0.0015%‐glucose, averaged over all spore bags with the same binned RNAP II level. n = 80 spore bags. Triangular relationship. Any pair of the following three are positively correlated: Germination ability for each glucose concentration (purple), GFP inducibility (green) and amounts of RNA polymerases I‐III. " width='250' height="auto" />

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    Images

    1) Product Images from "Dormancy‐to‐death transition in yeast spores occurs due to gradual loss of gene‐expressing ability"

    Article Title: Dormancy‐to‐death transition in yeast spores occurs due to gradual loss of gene‐expressing ability

    Journal: Molecular Systems Biology

    doi: 10.15252/msb.20199245

    More RNA polymerases I‐III means higher germination ability and GFP inducibility Protocol for (B). See
    Figure Legend Snippet: More RNA polymerases I‐III means higher germination ability and GFP inducibility Protocol for (B). See "Protocol for Fig 4B " in Materials and Methods. Thiolutin inhibits transcription. Cycloheximide inhibits translation. Antimycin A inhibits ATP synthesis by inhibiting oxidative phosphorylation. For experiment in (A), percentage of spore bags that germinated. n = 3; error bar are sem. Method to detect all RNAs being made in yeast spores with a single‐cell resolution. Spores were incubated for 24 h in PBS with 1 mM of 5‐Ethynyl Uridine (5‐EU; denoted U*) that incorporates into freshly made RNAs. We fix the spores afterwards and let fluorophore (Alexa 488) enter the spores and bind U* (see Materials and Methods). "Total RNA level" of a spore bag is th e total fluorescence from all the 5‐EU labelled RNAs. Microscope images show a spore bag (from strain “TS3”) after either incorporating 5‐EU as described in (C) (top two images) or, as a control, following the protocol in (C) but without the 5‐EU (bottom two images) (also see Appendix Fig S23 A). Scale bar: 2 μm. Top histogram: total RNA level per spore bag with 5‐EU (i.e. Alexa 488 fluorescence; n = 103 spore bags). Bottom histogram: fluorescence per spore bag in control population (i.e. without 5‐EU; n = 95 spore bags). Also see Appendix Fig S22 A. Each dot is from a single spore bag ("TS3" strain) with GFP‐inducing circuit (Fig 3A ). For each spore bag, we measured its GFP protein level ("GFP inducibility") and total RNA level (5‐EU fluorescence) after incubating the spores for 24 h in PBS with both 100‐μg/ml doxycycline and 1 mM of 5‐EU (see (C)). n = 245 spore bags. Alexa 594 fluorophore attached to 5‐EU (see Materials and Methods). Green line: linear regression with R = 0.24 and Pearson P ‐value = 0.00018. See "Protocol for Fig 4F " in Materials and Methods. GFP inducibility per spore bag (each dot, "TS8" strain) measured as in (E) but now with live time‐lapse without the 5‐EU. "RNAP II level" is the mCherry fluorescence per spore bag due to the mCherry protein fused to Rpb3, a subunit of RNA polymerase II. n = 182 spore bags; Red line: linear regression with R = 0.64 and Pearson P ‐value = 3.02 × 10 −22 . Scale bar = 2 μm. Same protocol as in (F) but with "TS9" spores. "RNAP I III level" is the mCherry fluorescence per spore bag due to the mCherry protein fused to Rpc40, a subunit of both RNAP I and RNAP III. n = 185 spore bags; Red line: linear regression with R = 0.63 and Pearson P ‐value = 6.6 × 10 −22 . Scale bar = 2 μm. Same protocol as in (F) but with "TT14" spores fixed after 24 h of incubation in PBS with 100‐μg/ml doxycycline. 18s rRNA level is from CAL Fluor Red 610 fluorescence emitted by single‐molecule FISH probes bound to 18S rRNAs (see Materials and Methods). n = 213 spore bags; Red line: linear regression with R = 0.19 and Pearson P ‐value = 0.005. Scale bar = 2 μm. Bottom: RNAP II levels of spore bags ("TS8" strain) in a population, measured as in (F). Top: As a function of the RNAP II level (binned in the histogram), percentage of spore bags that germinated after receiving a 0.0015%‐glucose, averaged over all spore bags with the same binned RNAP II level. n = 80 spore bags. Triangular relationship. Any pair of the following three are positively correlated: Germination ability for each glucose concentration (purple), GFP inducibility (green) and amounts of RNA polymerases I‐III.

    Techniques Used: Incubation, Fluorescence, Microscopy, Fluorescence In Situ Hybridization, Concentration Assay

    2) Product Images from "Unexpected diversity in eukaryotic transcription revealed by the retrotransposon hotspot family of Trypanosoma brucei"

    Article Title: Unexpected diversity in eukaryotic transcription revealed by the retrotransposon hotspot family of Trypanosoma brucei

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1255

    RHS are involved in global mRNA synthesis ( A ) Quantification of 5-ethynyl uridine (5-EU) incorporation. Each RNAi line was cultured in the presence or absence of tetracycline for 2 days and pulsed for 10 min with 5-EU ( n = 3). Values are given as a percentage of the uninduced control. P-values are shown for paired, one-tailed t-tests. Error bars = SD. ( B ) Correlation between reads per million (RPM) for nascent mRNAs from RNAi lines cultured in the presence or absence of tetracycline for 2 days. R: Pearson correlation coefficient. Biological replicates were performed. See also Supplemental Figure S5 .
    Figure Legend Snippet: RHS are involved in global mRNA synthesis ( A ) Quantification of 5-ethynyl uridine (5-EU) incorporation. Each RNAi line was cultured in the presence or absence of tetracycline for 2 days and pulsed for 10 min with 5-EU ( n = 3). Values are given as a percentage of the uninduced control. P-values are shown for paired, one-tailed t-tests. Error bars = SD. ( B ) Correlation between reads per million (RPM) for nascent mRNAs from RNAi lines cultured in the presence or absence of tetracycline for 2 days. R: Pearson correlation coefficient. Biological replicates were performed. See also Supplemental Figure S5 .

    Techniques Used: Cell Culture, One-tailed Test

    3) Product Images from "γH2AX in the S Phase After UV Irradiation Corresponds to the Sites of DNA Replication and Not DNA Damage"

    Article Title: γH2AX in the S Phase After UV Irradiation Corresponds to the Sites of DNA Replication and Not DNA Damage

    Journal: bioRxiv

    doi: 10.1101/810689

    UV-induced γH2AX does not report on the extent of DNA damage in the S phase cells (A) Definition of the colocalization metric: more than 50% overlap between the foci from the two channels at a position. Images are of NCS-treated cells: those 53BP1 foci colocalizing with γH2AX foci are considered as DSBs (B) Percentage of γH2AX foci which are double strand breaks for control, UV- and NCS-treated cells. For UV treatment a very little fraction of γH2AX foci in the S phase cells corresponds to DSBs. (C) Global transcription is measured in UV-treated cells by quantifying 5-ethynyl-uridine incorporation. (D) Bar graphs for γH2AX and EU are normalized across the four dosages. Resultant bar graphs for EU are inverted over those for γH2AX such that the total height of the two bars corresponding to control population in every cell cycle phase is unity. A clear gap is observed between γH2AX and EU bars for G1 and G2/M phases after UV treatment. The γH2AX bars in the S phase not just overlap with EU bars but goes past the unit box showing a disproportionate increase in γH2AX. (E) Mean levels of γH2AX increase with the increase in DNA damage as achieved by increasing the UV dosage in all the phases of the cell cycle. For all the dosages, there is a sharp γH2AX peak in the S phase. (F) Increase in DNA damage always leads to decrease in EU as seen across the population treated with different dosages of UV. But within a population there is no dip in EU levels corresponding to the γH2AX peak showing that γH2AX in the S phase after UV does not reflect on the total extent of DNA damage in those cells. Cells were treated with 10 J/m 2 and 1.6 µg/ml NCS for two minutes. Line graphs are showing relative levels with respect to the mean value of G1 phase in control cells. Scale bar: 10 µm.
    Figure Legend Snippet: UV-induced γH2AX does not report on the extent of DNA damage in the S phase cells (A) Definition of the colocalization metric: more than 50% overlap between the foci from the two channels at a position. Images are of NCS-treated cells: those 53BP1 foci colocalizing with γH2AX foci are considered as DSBs (B) Percentage of γH2AX foci which are double strand breaks for control, UV- and NCS-treated cells. For UV treatment a very little fraction of γH2AX foci in the S phase cells corresponds to DSBs. (C) Global transcription is measured in UV-treated cells by quantifying 5-ethynyl-uridine incorporation. (D) Bar graphs for γH2AX and EU are normalized across the four dosages. Resultant bar graphs for EU are inverted over those for γH2AX such that the total height of the two bars corresponding to control population in every cell cycle phase is unity. A clear gap is observed between γH2AX and EU bars for G1 and G2/M phases after UV treatment. The γH2AX bars in the S phase not just overlap with EU bars but goes past the unit box showing a disproportionate increase in γH2AX. (E) Mean levels of γH2AX increase with the increase in DNA damage as achieved by increasing the UV dosage in all the phases of the cell cycle. For all the dosages, there is a sharp γH2AX peak in the S phase. (F) Increase in DNA damage always leads to decrease in EU as seen across the population treated with different dosages of UV. But within a population there is no dip in EU levels corresponding to the γH2AX peak showing that γH2AX in the S phase after UV does not reflect on the total extent of DNA damage in those cells. Cells were treated with 10 J/m 2 and 1.6 µg/ml NCS for two minutes. Line graphs are showing relative levels with respect to the mean value of G1 phase in control cells. Scale bar: 10 µm.

    Techniques Used:

    4) Product Images from "Unexpected diversity in eukaryotic transcription revealed by the retrotransposon hotspot family of Trypanosoma brucei"

    Article Title: Unexpected diversity in eukaryotic transcription revealed by the retrotransposon hotspot family of Trypanosoma brucei

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1255

    RHS are involved in global mRNA synthesis ( A ) Quantification of 5-ethynyl uridine (5-EU) incorporation. Each RNAi line was cultured in the presence or absence of tetracycline for 2 days and pulsed for 10 min with 5-EU ( n = 3). Values are given as a percentage of the uninduced control. P-values are shown for paired, one-tailed t-tests. Error bars = SD. ( B ) Correlation between reads per million (RPM) for nascent mRNAs from RNAi lines cultured in the presence or absence of tetracycline for 2 days. R: Pearson correlation coefficient. Biological replicates were performed. See also Supplemental Figure S5 .
    Figure Legend Snippet: RHS are involved in global mRNA synthesis ( A ) Quantification of 5-ethynyl uridine (5-EU) incorporation. Each RNAi line was cultured in the presence or absence of tetracycline for 2 days and pulsed for 10 min with 5-EU ( n = 3). Values are given as a percentage of the uninduced control. P-values are shown for paired, one-tailed t-tests. Error bars = SD. ( B ) Correlation between reads per million (RPM) for nascent mRNAs from RNAi lines cultured in the presence or absence of tetracycline for 2 days. R: Pearson correlation coefficient. Biological replicates were performed. See also Supplemental Figure S5 .

    Techniques Used: Cell Culture, One-tailed Test

    5) Product Images from "γH2AX in the S Phase After UV Irradiation Corresponds to the Sites of DNA Replication and Not DNA Damage"

    Article Title: γH2AX in the S Phase After UV Irradiation Corresponds to the Sites of DNA Replication and Not DNA Damage

    Journal: bioRxiv

    doi: 10.1101/810689

    UV-induced γH2AX does not report on the extent of DNA damage in the S phase cells (A) Definition of the colocalization metric: more than 50% overlap between the foci from the two channels at a position. Images are of NCS-treated cells: those 53BP1 foci colocalizing with γH2AX foci are considered as DSBs (B) Percentage of γH2AX foci which are double strand breaks for control, UV- and NCS-treated cells. For UV treatment a very little fraction of γH2AX foci in the S phase cells corresponds to DSBs. (C) Global transcription is measured in UV-treated cells by quantifying 5-ethynyl-uridine incorporation. (D) Bar graphs for γH2AX and EU are normalized across the four dosages. Resultant bar graphs for EU are inverted over those for γH2AX such that the total height of the two bars corresponding to control population in every cell cycle phase is unity. A clear gap is observed between γH2AX and EU bars for G1 and G2/M phases after UV treatment. The γH2AX bars in the S phase not just overlap with EU bars but goes past the unit box showing a disproportionate increase in γH2AX. (E) Mean levels of γH2AX increase with the increase in DNA damage as achieved by increasing the UV dosage in all the phases of the cell cycle. For all the dosages, there is a sharp γH2AX peak in the S phase. (F) Increase in DNA damage always leads to decrease in EU as seen across the population treated with different dosages of UV. But within a population there is no dip in EU levels corresponding to the γH2AX peak showing that γH2AX in the S phase after UV does not reflect on the total extent of DNA damage in those cells. Cells were treated with 10 J/m 2 and 1.6 µg/ml NCS for two minutes. Line graphs are showing relative levels with respect to the mean value of G1 phase in control cells. Scale bar: 10 µm.
    Figure Legend Snippet: UV-induced γH2AX does not report on the extent of DNA damage in the S phase cells (A) Definition of the colocalization metric: more than 50% overlap between the foci from the two channels at a position. Images are of NCS-treated cells: those 53BP1 foci colocalizing with γH2AX foci are considered as DSBs (B) Percentage of γH2AX foci which are double strand breaks for control, UV- and NCS-treated cells. For UV treatment a very little fraction of γH2AX foci in the S phase cells corresponds to DSBs. (C) Global transcription is measured in UV-treated cells by quantifying 5-ethynyl-uridine incorporation. (D) Bar graphs for γH2AX and EU are normalized across the four dosages. Resultant bar graphs for EU are inverted over those for γH2AX such that the total height of the two bars corresponding to control population in every cell cycle phase is unity. A clear gap is observed between γH2AX and EU bars for G1 and G2/M phases after UV treatment. The γH2AX bars in the S phase not just overlap with EU bars but goes past the unit box showing a disproportionate increase in γH2AX. (E) Mean levels of γH2AX increase with the increase in DNA damage as achieved by increasing the UV dosage in all the phases of the cell cycle. For all the dosages, there is a sharp γH2AX peak in the S phase. (F) Increase in DNA damage always leads to decrease in EU as seen across the population treated with different dosages of UV. But within a population there is no dip in EU levels corresponding to the γH2AX peak showing that γH2AX in the S phase after UV does not reflect on the total extent of DNA damage in those cells. Cells were treated with 10 J/m 2 and 1.6 µg/ml NCS for two minutes. Line graphs are showing relative levels with respect to the mean value of G1 phase in control cells. Scale bar: 10 µm.

    Techniques Used:

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    Hybridization:

    Article Title: The Dynamics of Cytoplasmic mRNA Metabolism.
    Article Snippet: STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d KEY RESOURCES TABLE d LEAD CONTACT AND MATERIALS AVAILABILITY d EXPERIMENTAL MODEL AND SUBJECT DETAILS 12 B Cell Lines and Cell Culture d METHOD DETAILS B Metabolic-Labeling Time Courses B RNA Standards B Biotinylation of 5EU Labeled RNA B Purification of Biotinylated RNA B PAL-Seq v2 B PAL-Seq v2 Data Analysis B Analysis of PAL-Seq ss-Ligation Data B TAIL-Seq B RNA-Seq B Calculation of mRNA Half-Lives B Model of mRNA Metabolism B Bootstrap Analysis B Background Subtraction for PAL-Seq Data B ActD Treatment Molecular Cell 77, 1–14, February 20, 2020 d QUANTIFICATION AND STATISTICAL ANALYSIS d DATA AND CODE AVAILABILITY SUPPLEMENTAL INFORMATION Supplemental Information can be found online at https://doi.org/10.1016/j. molcel.2019.12.005. .. KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, Peptides, and Recombinant Proteins cOmplete Mini EDTA-free Protease Inhibitor tablets Roche Cat# 4693159001 TRI Reagent Ambion Cat# AM9738 Bovine Calf Serum Sigma Cat# 12133C 5-ethynyluridine Jena Biosciences Cat# CLK-N002-10 Vaccinia capping system NEB Cat# M2080S SUPERaseIn Thermo Fisher Cat# AM2694 T4 polynucleotide kinase NEB Cat# M0201S MEGAscript T7 Thermo Fisher Cat# AM1333 5-ethynyluridine-triphosphate Jena Biosciences Cat# CLK-T08 Oligo(dT) Dynabeads Thermo Fisher Cat# 61002 Disulfide biotin azide Click Chemistry Tools Cat# 1168-10 Tris(3-hydroxypropyltriazolylmethyl)amine Click Chemistry Tools Cat# 1010-100 Sodium Ascorbate Sigma Cat# 11140 Dynabeads MyOne Streptavidin C1 beads Thermo Fisher Cat# 65001 Yeast total RNA Thermo Fisher Cat# AM7118 Tris(2-carboxyethyl)phosphine Sigma Cat# 646547 RNase T1 Thermo Fisher Cat# AM2283 Superscript III Reverse Transcriptase Invitrogen Cat# 18080044 Titanium Taq Polymerase Takara Cat# 639208 AMPure beads Beckman Coulter Cat# NC9933872 RiboZero Gold HMR Illumina Cat# MRZG12324 T4 RNA ligase I NEB Cat# M0437 T4 RNA ligase II, truncated KQ NEB Cat# M0373 HT2 hybridization buffer Illumina HT2 dTTP Thermo Fisher Cat# R0171 [g-32P]ATP Perkin Elmer Cat# NEG035C001MC [a-32P]GTP Perkin Elmer Cat# NEG006H500UC Critical Commercial Assays Micro Bio-Spin P30 gel columns Bio-Rad 7326250 Deposited Data Fragmented RNAseq This study GEO: GSE134660 PAL-seq This study GEO: GSE134660 Experimental Models: Cell Lines Mouse: miR-1–inducible 3T3 cell line Eichhorn et al., 2014 N/A Mouse: miR-155–inducible 3T3 cell line Eichhorn et al., 2014 N/A Oligonucleotides See Table S3 This paper N/A Library preparation adapters and primers (also listed in Table S3) IDT N/A RNA standards (also listed in Table S3) Subtelny et al., 2014 N/A (Continued on next page) Molecular Cell 77, 1–14.e1–e10, February 20, 2020 e1 .. Continued REAGENT or RESOURCE SOURCE IDENTIFIER Software and Algorithms R 3.6.1 The R Foundation for Statistical Computing https://www.r-project.org/foundation/ Python The Python Software Foundation https://www.python.org/; RRID: SCR_008394 Matplotlib 2.0.0 Hunter, 2007 https://matplotlib.org/; RRID: SCR_008624 Numpy 1.14.3 Oliphant, 2007 https://numpy.org; RRID: SCR_008633 Scipy 1.0.1 Oliphant, 2007 https://www.scipy.org; RRID: SCR_008058 scikit-learn 0.18.1 Pedregosa et al., 2011 https://scikit-learn.org/stable/; RRID: SCR_002577 Hmmlearn 0.2.0 Hmmlearn developers, 2010 https://hmmlearn.readthedocs.io/en/latest/ Ghmm Alexander Schliep http://ghmm.org/ STAR v2.5.4b Dobin et al., 2013 RRID: SCR_015899 Bedtools v2.26.0 Quinlan and Hall, 2010 RRID: SCR_006646 Samtools Li et al., 2009 RRID: SCR_002105 Tail kinetics model This study https://github.com/timeisen/ DynamicsOfCytoplasmicMrnaMetabolism Exponential decay model This study https://github.com/timeisen/ DynamicsOfCytoplasmicMrnaMetabolism Tail length determination for splintligation data This study https://github.com/kslin/PAL-seq Tail length determination for singlestranded ligation data This study https://github.com/kslin/PAL-seq Flow cell configuration for a HiSeq2500 running PALseq This study https://github.com/kslin/PAL-seq L-BFGS-B optimization algorithm Nocedal, 1980 http://www.uivt.cas.cz/ luksan/ subroutines.html deSolve v1.21 Soetaert et al., 2010 https://cran.r-project.org/web/packages/ deSolve/citation.html NLopt v1.0.4 Steven G. Johnson, The NLopt nonlinear-optimization package. https://nlopt.readthedocs.io/en/latest/ Tidyverse v1.2.1 Wickham et al., 2019 https://www.tidyverse.org/

    Polyacrylamide Gel Electrophoresis:

    Article Title: The Dynamics of Cytoplasmic mRNA Metabolism.
    Article Snippet: STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d KEY RESOURCES TABLE d LEAD CONTACT AND MATERIALS AVAILABILITY d EXPERIMENTAL MODEL AND SUBJECT DETAILS 12 B Cell Lines and Cell Culture d METHOD DETAILS B Metabolic-Labeling Time Courses B RNA Standards B Biotinylation of 5EU Labeled RNA B Purification of Biotinylated RNA B PAL-Seq v2 B PAL-Seq v2 Data Analysis B Analysis of PAL-Seq ss-Ligation Data B TAIL-Seq B RNA-Seq B Calculation of mRNA Half-Lives B Model of mRNA Metabolism B Bootstrap Analysis B Background Subtraction for PAL-Seq Data B ActD Treatment Molecular Cell 77, 1–14, February 20, 2020 d QUANTIFICATION AND STATISTICAL ANALYSIS d DATA AND CODE AVAILABILITY SUPPLEMENTAL INFORMATION Supplemental Information can be found online at https://doi.org/10.1016/j. molcel.2019.12.005. .. KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, Peptides, and Recombinant Proteins cOmplete Mini EDTA-free Protease Inhibitor tablets Roche Cat# 4693159001 TRI Reagent Ambion Cat# AM9738 Bovine Calf Serum Sigma Cat# 12133C 5-ethynyluridine Jena Biosciences Cat# CLK-N002-10 Vaccinia capping system NEB Cat# M2080S SUPERaseIn Thermo Fisher Cat# AM2694 T4 polynucleotide kinase NEB Cat# M0201S MEGAscript T7 Thermo Fisher Cat# AM1333 5-ethynyluridine-triphosphate Jena Biosciences Cat# CLK-T08 Oligo(dT) Dynabeads Thermo Fisher Cat# 61002 Disulfide biotin azide Click Chemistry Tools Cat# 1168-10 Tris(3-hydroxypropyltriazolylmethyl)amine Click Chemistry Tools Cat# 1010-100 Sodium Ascorbate Sigma Cat# 11140 Dynabeads MyOne Streptavidin C1 beads Thermo Fisher Cat# 65001 Yeast total RNA Thermo Fisher Cat# AM7118 Tris(2-carboxyethyl)phosphine Sigma Cat# 646547 RNase T1 Thermo Fisher Cat# AM2283 Superscript III Reverse Transcriptase Invitrogen Cat# 18080044 Titanium Taq Polymerase Takara Cat# 639208 AMPure beads Beckman Coulter Cat# NC9933872 RiboZero Gold HMR Illumina Cat# MRZG12324 T4 RNA ligase I NEB Cat# M0437 T4 RNA ligase II, truncated KQ NEB Cat# M0373 HT2 hybridization buffer Illumina HT2 dTTP Thermo Fisher Cat# R0171 [g-32P]ATP Perkin Elmer Cat# NEG035C001MC [a-32P]GTP Perkin Elmer Cat# NEG006H500UC Critical Commercial Assays Micro Bio-Spin P30 gel columns Bio-Rad 7326250 Deposited Data Fragmented RNAseq This study GEO: GSE134660 PAL-seq This study GEO: GSE134660 Experimental Models: Cell Lines Mouse: miR-1–inducible 3T3 cell line Eichhorn et al., 2014 N/A Mouse: miR-155–inducible 3T3 cell line Eichhorn et al., 2014 N/A Oligonucleotides See Table S3 This paper N/A Library preparation adapters and primers (also listed in Table S3) IDT N/A RNA standards (also listed in Table S3) Subtelny et al., 2014 N/A (Continued on next page) Molecular Cell 77, 1–14.e1–e10, February 20, 2020 e1 .. Continued REAGENT or RESOURCE SOURCE IDENTIFIER Software and Algorithms R 3.6.1 The R Foundation for Statistical Computing https://www.r-project.org/foundation/ Python The Python Software Foundation https://www.python.org/; RRID: SCR_008394 Matplotlib 2.0.0 Hunter, 2007 https://matplotlib.org/; RRID: SCR_008624 Numpy 1.14.3 Oliphant, 2007 https://numpy.org; RRID: SCR_008633 Scipy 1.0.1 Oliphant, 2007 https://www.scipy.org; RRID: SCR_008058 scikit-learn 0.18.1 Pedregosa et al., 2011 https://scikit-learn.org/stable/; RRID: SCR_002577 Hmmlearn 0.2.0 Hmmlearn developers, 2010 https://hmmlearn.readthedocs.io/en/latest/ Ghmm Alexander Schliep http://ghmm.org/ STAR v2.5.4b Dobin et al., 2013 RRID: SCR_015899 Bedtools v2.26.0 Quinlan and Hall, 2010 RRID: SCR_006646 Samtools Li et al., 2009 RRID: SCR_002105 Tail kinetics model This study https://github.com/timeisen/ DynamicsOfCytoplasmicMrnaMetabolism Exponential decay model This study https://github.com/timeisen/ DynamicsOfCytoplasmicMrnaMetabolism Tail length determination for splintligation data This study https://github.com/kslin/PAL-seq Tail length determination for singlestranded ligation data This study https://github.com/kslin/PAL-seq Flow cell configuration for a HiSeq2500 running PALseq This study https://github.com/kslin/PAL-seq L-BFGS-B optimization algorithm Nocedal, 1980 http://www.uivt.cas.cz/ luksan/ subroutines.html deSolve v1.21 Soetaert et al., 2010 https://cran.r-project.org/web/packages/ deSolve/citation.html NLopt v1.0.4 Steven G. Johnson, The NLopt nonlinear-optimization package. https://nlopt.readthedocs.io/en/latest/ Tidyverse v1.2.1 Wickham et al., 2019 https://www.tidyverse.org/

    other:

    Article Title: Visualization of the Nucleolus Using Ethynyl Uridine
    Article Snippet: When CLK-N002-10 product was used, 20 μl of DMSO was added to the media to keep the same conditions as for the E-10345 product.

    Synthesized:

    Article Title: Dormancy‐to‐death transition in yeast spores occurs due to gradual loss of gene‐expressing ability
    Article Snippet: Single‐molecule RNA FISH We used the standard protocol for single‐molecule RNA FISH in yeast, detailed in “Protocol for S.cerevisiae from Stellaris RNA FISH” (Biosearch Technologies, Inc., Petaluma, CA) and also in Raj et al ( ) and Youk et al ( ). .. Metabolic (5‐Ethynyl Uridine) labelling of freshly synthesized RNA with click reaction We used click chemistry to bind fluorophores to the 5‐Ethynyl Uridine (5‐EU) labelled RNAs in spores. .. To do so, we followed the protocol for mammalian cells, from “Click‐iT® Plus Alexa Fluor® picolyl azide toolkits” (Thermo Fisher) but with the following modifications so that the protocol would work in yeast spores: (i) all reactions and washing steps were done in a 1.5‐ml Eppendorf tube with a centrifugation speed of 800 g ; (ii) spore fixation and permeabilization were performed as mentioned above; (iii) total volume for the click reaction was 50 μl; (iv) for the click reaction, fixed spores were at OD ~ 0.02; (v) the click‐reaction cocktail was incubated for 1 h and 30 min. All components (Ethynyl‐Uridine, Fluorophore‐Azide and reagents for the click reaction) were from the "Click‐iT® Plus Alexa Fluor® picolyl azide toolkits" (Thermo Fisher).

    Irradiation:

    Article Title: γH2AX in the S Phase After UV Irradiation Corresponds to the Sites of DNA Replication and Not DNA Damage
    Article Snippet: .. For measuring global transcription, cells were labelled with 5-Ethynyl-Uridine (EU) immediately after UV irradiation for 30 minutes. .. EU gets incorporated into actively transcribing RNAs and marks the translational activity inside the cells which can later be detected using click chemistry.

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    Jena Bioscience 5 ethynyl uridine
    More RNA polymerases I‐III means higher germination ability and GFP inducibility Protocol for (B). See 5‐Ethynyl Uridine (5‐EU; denoted U*) that incorporates into freshly made RNAs. We fix the spores afterwards and let fluorophore (Alexa 488) enter the spores and bind U* (see Materials and Methods). "Total RNA level" of a spore bag is th e total fluorescence from all the 5‐EU labelled RNAs. Microscope images show a spore bag (from strain “TS3”) after either incorporating 5‐EU as described in (C) (top two images) or, as a control, following the protocol in (C) but without the 5‐EU (bottom two images) (also see Appendix Fig S23 A). Scale bar: 2 μm. Top histogram: total RNA level per spore bag with 5‐EU (i.e. Alexa 488 fluorescence; n = 103 spore bags). Bottom histogram: fluorescence per spore bag in control population (i.e. without 5‐EU; n = 95 spore bags). Also see Appendix Fig S22 A. Each dot is from a single spore bag ("TS3" strain) with GFP‐inducing circuit (Fig 3A ). For each spore bag, we measured its GFP protein level ("GFP inducibility") and total RNA level (5‐EU fluorescence) after incubating the spores for 24 h in PBS with both 100‐μg/ml doxycycline and 1 mM of 5‐EU (see (C)). n = 245 spore bags. Alexa 594 fluorophore attached to 5‐EU (see Materials and Methods). Green line: linear regression with R = 0.24 and Pearson P ‐value = 0.00018. See "Protocol for Fig 4F " in Materials and Methods. GFP inducibility per spore bag (each dot, "TS8" strain) measured as in (E) but now with live time‐lapse without the 5‐EU. "RNAP II level" is the mCherry fluorescence per spore bag due to the mCherry protein fused to Rpb3, a subunit of RNA polymerase II. n = 182 spore bags; Red line: linear regression with R = 0.64 and Pearson P ‐value = 3.02 × 10 −22 . Scale bar = 2 μm. Same protocol as in (F) but with "TS9" spores. "RNAP I III level" is the mCherry fluorescence per spore bag due to the mCherry protein fused to Rpc40, a subunit of both RNAP I and RNAP III. n = 185 spore bags; Red line: linear regression with R = 0.63 and Pearson P ‐value = 6.6 × 10 −22 . Scale bar = 2 μm. Same protocol as in (F) but with "TT14" spores fixed after 24 h of incubation in PBS with 100‐μg/ml doxycycline. 18s rRNA level is from CAL Fluor Red 610 fluorescence emitted by single‐molecule FISH probes bound to 18S rRNAs (see Materials and Methods). n = 213 spore bags; Red line: linear regression with R = 0.19 and Pearson P ‐value = 0.005. Scale bar = 2 μm. Bottom: RNAP II levels of spore bags ("TS8" strain) in a population, measured as in (F). Top: As a function of the RNAP II level (binned in the histogram), percentage of spore bags that germinated after receiving a 0.0015%‐glucose, averaged over all spore bags with the same binned RNAP II level. n = 80 spore bags. Triangular relationship. Any pair of the following three are positively correlated: Germination ability for each glucose concentration (purple), GFP inducibility (green) and amounts of RNA polymerases I‐III. " width="250" height="auto" />
    5 Ethynyl Uridine, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    More RNA polymerases I‐III means higher germination ability and GFP inducibility Protocol for (B). See

    Journal: Molecular Systems Biology

    Article Title: Dormancy‐to‐death transition in yeast spores occurs due to gradual loss of gene‐expressing ability

    doi: 10.15252/msb.20199245

    Figure Lengend Snippet: More RNA polymerases I‐III means higher germination ability and GFP inducibility Protocol for (B). See "Protocol for Fig 4B " in Materials and Methods. Thiolutin inhibits transcription. Cycloheximide inhibits translation. Antimycin A inhibits ATP synthesis by inhibiting oxidative phosphorylation. For experiment in (A), percentage of spore bags that germinated. n = 3; error bar are sem. Method to detect all RNAs being made in yeast spores with a single‐cell resolution. Spores were incubated for 24 h in PBS with 1 mM of 5‐Ethynyl Uridine (5‐EU; denoted U*) that incorporates into freshly made RNAs. We fix the spores afterwards and let fluorophore (Alexa 488) enter the spores and bind U* (see Materials and Methods). "Total RNA level" of a spore bag is th e total fluorescence from all the 5‐EU labelled RNAs. Microscope images show a spore bag (from strain “TS3”) after either incorporating 5‐EU as described in (C) (top two images) or, as a control, following the protocol in (C) but without the 5‐EU (bottom two images) (also see Appendix Fig S23 A). Scale bar: 2 μm. Top histogram: total RNA level per spore bag with 5‐EU (i.e. Alexa 488 fluorescence; n = 103 spore bags). Bottom histogram: fluorescence per spore bag in control population (i.e. without 5‐EU; n = 95 spore bags). Also see Appendix Fig S22 A. Each dot is from a single spore bag ("TS3" strain) with GFP‐inducing circuit (Fig 3A ). For each spore bag, we measured its GFP protein level ("GFP inducibility") and total RNA level (5‐EU fluorescence) after incubating the spores for 24 h in PBS with both 100‐μg/ml doxycycline and 1 mM of 5‐EU (see (C)). n = 245 spore bags. Alexa 594 fluorophore attached to 5‐EU (see Materials and Methods). Green line: linear regression with R = 0.24 and Pearson P ‐value = 0.00018. See "Protocol for Fig 4F " in Materials and Methods. GFP inducibility per spore bag (each dot, "TS8" strain) measured as in (E) but now with live time‐lapse without the 5‐EU. "RNAP II level" is the mCherry fluorescence per spore bag due to the mCherry protein fused to Rpb3, a subunit of RNA polymerase II. n = 182 spore bags; Red line: linear regression with R = 0.64 and Pearson P ‐value = 3.02 × 10 −22 . Scale bar = 2 μm. Same protocol as in (F) but with "TS9" spores. "RNAP I III level" is the mCherry fluorescence per spore bag due to the mCherry protein fused to Rpc40, a subunit of both RNAP I and RNAP III. n = 185 spore bags; Red line: linear regression with R = 0.63 and Pearson P ‐value = 6.6 × 10 −22 . Scale bar = 2 μm. Same protocol as in (F) but with "TT14" spores fixed after 24 h of incubation in PBS with 100‐μg/ml doxycycline. 18s rRNA level is from CAL Fluor Red 610 fluorescence emitted by single‐molecule FISH probes bound to 18S rRNAs (see Materials and Methods). n = 213 spore bags; Red line: linear regression with R = 0.19 and Pearson P ‐value = 0.005. Scale bar = 2 μm. Bottom: RNAP II levels of spore bags ("TS8" strain) in a population, measured as in (F). Top: As a function of the RNAP II level (binned in the histogram), percentage of spore bags that germinated after receiving a 0.0015%‐glucose, averaged over all spore bags with the same binned RNAP II level. n = 80 spore bags. Triangular relationship. Any pair of the following three are positively correlated: Germination ability for each glucose concentration (purple), GFP inducibility (green) and amounts of RNA polymerases I‐III.

    Article Snippet: Metabolic (5‐Ethynyl Uridine) labelling of freshly synthesized RNA with click reaction We used click chemistry to bind fluorophores to the 5‐Ethynyl Uridine (5‐EU) labelled RNAs in spores.

    Techniques: Incubation, Fluorescence, Microscopy, Fluorescence In Situ Hybridization, Concentration Assay

    RHS are involved in global mRNA synthesis ( A ) Quantification of 5-ethynyl uridine (5-EU) incorporation. Each RNAi line was cultured in the presence or absence of tetracycline for 2 days and pulsed for 10 min with 5-EU ( n = 3). Values are given as a percentage of the uninduced control. P-values are shown for paired, one-tailed t-tests. Error bars = SD. ( B ) Correlation between reads per million (RPM) for nascent mRNAs from RNAi lines cultured in the presence or absence of tetracycline for 2 days. R: Pearson correlation coefficient. Biological replicates were performed. See also Supplemental Figure S5 .

    Journal: Nucleic Acids Research

    Article Title: Unexpected diversity in eukaryotic transcription revealed by the retrotransposon hotspot family of Trypanosoma brucei

    doi: 10.1093/nar/gky1255

    Figure Lengend Snippet: RHS are involved in global mRNA synthesis ( A ) Quantification of 5-ethynyl uridine (5-EU) incorporation. Each RNAi line was cultured in the presence or absence of tetracycline for 2 days and pulsed for 10 min with 5-EU ( n = 3). Values are given as a percentage of the uninduced control. P-values are shown for paired, one-tailed t-tests. Error bars = SD. ( B ) Correlation between reads per million (RPM) for nascent mRNAs from RNAi lines cultured in the presence or absence of tetracycline for 2 days. R: Pearson correlation coefficient. Biological replicates were performed. See also Supplemental Figure S5 .

    Article Snippet: 5-Ethynyl uridine incorporation and processing for GRO-Seq Procyclic forms at a density of 7–8 × 106 ml−1 were pulsed for 10 min with 200 μM 5-ethynyl uridine (5-EU; Jena Biosciences).

    Techniques: Cell Culture, One-tailed Test

    UV-induced γH2AX does not report on the extent of DNA damage in the S phase cells (A) Definition of the colocalization metric: more than 50% overlap between the foci from the two channels at a position. Images are of NCS-treated cells: those 53BP1 foci colocalizing with γH2AX foci are considered as DSBs (B) Percentage of γH2AX foci which are double strand breaks for control, UV- and NCS-treated cells. For UV treatment a very little fraction of γH2AX foci in the S phase cells corresponds to DSBs. (C) Global transcription is measured in UV-treated cells by quantifying 5-ethynyl-uridine incorporation. (D) Bar graphs for γH2AX and EU are normalized across the four dosages. Resultant bar graphs for EU are inverted over those for γH2AX such that the total height of the two bars corresponding to control population in every cell cycle phase is unity. A clear gap is observed between γH2AX and EU bars for G1 and G2/M phases after UV treatment. The γH2AX bars in the S phase not just overlap with EU bars but goes past the unit box showing a disproportionate increase in γH2AX. (E) Mean levels of γH2AX increase with the increase in DNA damage as achieved by increasing the UV dosage in all the phases of the cell cycle. For all the dosages, there is a sharp γH2AX peak in the S phase. (F) Increase in DNA damage always leads to decrease in EU as seen across the population treated with different dosages of UV. But within a population there is no dip in EU levels corresponding to the γH2AX peak showing that γH2AX in the S phase after UV does not reflect on the total extent of DNA damage in those cells. Cells were treated with 10 J/m 2 and 1.6 µg/ml NCS for two minutes. Line graphs are showing relative levels with respect to the mean value of G1 phase in control cells. Scale bar: 10 µm.

    Journal: bioRxiv

    Article Title: γH2AX in the S Phase After UV Irradiation Corresponds to the Sites of DNA Replication and Not DNA Damage

    doi: 10.1101/810689

    Figure Lengend Snippet: UV-induced γH2AX does not report on the extent of DNA damage in the S phase cells (A) Definition of the colocalization metric: more than 50% overlap between the foci from the two channels at a position. Images are of NCS-treated cells: those 53BP1 foci colocalizing with γH2AX foci are considered as DSBs (B) Percentage of γH2AX foci which are double strand breaks for control, UV- and NCS-treated cells. For UV treatment a very little fraction of γH2AX foci in the S phase cells corresponds to DSBs. (C) Global transcription is measured in UV-treated cells by quantifying 5-ethynyl-uridine incorporation. (D) Bar graphs for γH2AX and EU are normalized across the four dosages. Resultant bar graphs for EU are inverted over those for γH2AX such that the total height of the two bars corresponding to control population in every cell cycle phase is unity. A clear gap is observed between γH2AX and EU bars for G1 and G2/M phases after UV treatment. The γH2AX bars in the S phase not just overlap with EU bars but goes past the unit box showing a disproportionate increase in γH2AX. (E) Mean levels of γH2AX increase with the increase in DNA damage as achieved by increasing the UV dosage in all the phases of the cell cycle. For all the dosages, there is a sharp γH2AX peak in the S phase. (F) Increase in DNA damage always leads to decrease in EU as seen across the population treated with different dosages of UV. But within a population there is no dip in EU levels corresponding to the γH2AX peak showing that γH2AX in the S phase after UV does not reflect on the total extent of DNA damage in those cells. Cells were treated with 10 J/m 2 and 1.6 µg/ml NCS for two minutes. Line graphs are showing relative levels with respect to the mean value of G1 phase in control cells. Scale bar: 10 µm.

    Article Snippet: EU (CLK-N002-10) and EdU (CLK-N001-100) were procured from Jena Bioscience.

    Techniques: