t4 rna ligase buffer  (New England Biolabs)


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    T4 RNA Ligase Reaction Buffer
    Description:
    T4 RNA Ligase Reaction Buffer 3 0 ml
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    b0216l
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    3 0 ml
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    New England Biolabs t4 rna ligase buffer
    T4 RNA Ligase Reaction Buffer
    T4 RNA Ligase Reaction Buffer 3 0 ml
    https://www.bioz.com/result/t4 rna ligase buffer/product/New England Biolabs
    Average 99 stars, based on 274 article reviews
    Price from $9.99 to $1999.99
    t4 rna ligase buffer - by Bioz Stars, 2020-08
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    Images

    1) Product Images from "Strand-specific deep sequencing of the transcriptome"

    Article Title: Strand-specific deep sequencing of the transcriptome

    Journal: Genome Research

    doi: 10.1101/gr.094318.109

    DSSS protocol workflow. ( A ) Fragmentation. RNA is fragmented to sizes in the range of 60–200 nt. ( B ) Dephosphorylation. 5′ phosphates are removed from RNA by treatment with alkaline phosphatase. ( C ) 3′ adapter ligation. Dephosphorylated 200-nt-long RNA fragments are selected by urea-PAGE. The 3′ adapter is ligated to the 3′ ends using T4 RNA ligase I. ( D ) Rephosphorylation. Fragments are rephosphorylated by treatment with T4 polynucleotide kinase as preparation for the next ligation step. ( E ) 5′ adapter ligation, preceded by removal of the nonligated 3′adapter by urea-PAGE size selection. ( F ) Reverse transcription (RT) and amplification of library. Molecules with 5′ and 3′ adapters were selected by urea-PAGE. First strand cDNA synthesis and PCR amplification were carried out with the indicated primers. ( G ) Sequencing.
    Figure Legend Snippet: DSSS protocol workflow. ( A ) Fragmentation. RNA is fragmented to sizes in the range of 60–200 nt. ( B ) Dephosphorylation. 5′ phosphates are removed from RNA by treatment with alkaline phosphatase. ( C ) 3′ adapter ligation. Dephosphorylated 200-nt-long RNA fragments are selected by urea-PAGE. The 3′ adapter is ligated to the 3′ ends using T4 RNA ligase I. ( D ) Rephosphorylation. Fragments are rephosphorylated by treatment with T4 polynucleotide kinase as preparation for the next ligation step. ( E ) 5′ adapter ligation, preceded by removal of the nonligated 3′adapter by urea-PAGE size selection. ( F ) Reverse transcription (RT) and amplification of library. Molecules with 5′ and 3′ adapters were selected by urea-PAGE. First strand cDNA synthesis and PCR amplification were carried out with the indicated primers. ( G ) Sequencing.

    Techniques Used: De-Phosphorylation Assay, Ligation, Polyacrylamide Gel Electrophoresis, Selection, Amplification, Polymerase Chain Reaction, Sequencing

    2) Product Images from "Late steps of ribosome assembly in E. coli are sensitive to a severe heat stress but are assisted by the HSP70 chaperone machine †"

    Article Title: Late steps of ribosome assembly in E. coli are sensitive to a severe heat stress but are assisted by the HSP70 chaperone machine †

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq1049

    ( A ) Schematic processing of the p1 16S rRNA. The extra-sequences of 115 nt and 33 nt, flanking the m 16S rRNA at its 5′ and 3′ ends, respectively, are shown on a grey background. RA and FA are the primers used for 3′5′ RACE analysis. The site of annealing of RA to m 16S rRNA, and that of FA to the reverse complement of the m 16S rRNA, are indicated by arrows. Figure not drawn to scale. ( B ) Expected sizes in bp of the RT-PCR products (amplicons) obtained from the different species of 16S rRNA ( p1 , p2 , p3 and m ) by 3′5′ RACE. ( C–F ) Agarose gel electrophoresis of RT-PCR products obtained by 3′5′ RACE from total RNA isolated from MC4100 bacteria grown at 30°C (C), or 44°C (D), or 45°C (E) or 46°C (F). Each RNA sample was thermo-denatured (lanes b), or not (lanes a) prior to the 3′5′ ligation. The sizes (in bp) of the molecular weight markers are indicated to the left of each gel (M). ( G ) The thermodenaturation step dissociates the complementary sequences present at the 3′ and 5′ends of the p1 16S rRNA, and therefore offers to all the 16S rRNA species an equal chance to access to the T4 RNA ligase.
    Figure Legend Snippet: ( A ) Schematic processing of the p1 16S rRNA. The extra-sequences of 115 nt and 33 nt, flanking the m 16S rRNA at its 5′ and 3′ ends, respectively, are shown on a grey background. RA and FA are the primers used for 3′5′ RACE analysis. The site of annealing of RA to m 16S rRNA, and that of FA to the reverse complement of the m 16S rRNA, are indicated by arrows. Figure not drawn to scale. ( B ) Expected sizes in bp of the RT-PCR products (amplicons) obtained from the different species of 16S rRNA ( p1 , p2 , p3 and m ) by 3′5′ RACE. ( C–F ) Agarose gel electrophoresis of RT-PCR products obtained by 3′5′ RACE from total RNA isolated from MC4100 bacteria grown at 30°C (C), or 44°C (D), or 45°C (E) or 46°C (F). Each RNA sample was thermo-denatured (lanes b), or not (lanes a) prior to the 3′5′ ligation. The sizes (in bp) of the molecular weight markers are indicated to the left of each gel (M). ( G ) The thermodenaturation step dissociates the complementary sequences present at the 3′ and 5′ends of the p1 16S rRNA, and therefore offers to all the 16S rRNA species an equal chance to access to the T4 RNA ligase.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Isolation, Ligation, Molecular Weight

    3) Product Images from "Loss of a Universal tRNA Feature ▿"

    Article Title: Loss of a Universal tRNA Feature ▿

    Journal:

    doi: 10.1128/JB.01203-06

    tRNA end sequence analysis. A-C. RNA ligation products. Joints formed by T4 RNA ligase (brackets), involving the circled 5′-monophosphate ends, as revealed by RT-PCR with the indicated primers. Sequences of precursor RNA sequences are shown with
    Figure Legend Snippet: tRNA end sequence analysis. A-C. RNA ligation products. Joints formed by T4 RNA ligase (brackets), involving the circled 5′-monophosphate ends, as revealed by RT-PCR with the indicated primers. Sequences of precursor RNA sequences are shown with

    Techniques Used: Sequencing, Ligation, Reverse Transcription Polymerase Chain Reaction

    4) Product Images from "TSS-EMOTE, a refined protocol for a more complete and less biased global mapping of transcription start sites in bacterial pathogens"

    Article Title: TSS-EMOTE, a refined protocol for a more complete and less biased global mapping of transcription start sites in bacterial pathogens

    Journal: BMC Genomics

    doi: 10.1186/s12864-016-3211-3

    TSS-EMOTE flowchart. The TSS-EMOTE assay consists of a wet-lab library preparation (panels a to g ) and in silico analyses (panel H to N). An asterisk continually marks the original 5’-base of tri-phosphorylated RNA (thin red line). a Total RNA is purified, and digested with XRN1 5’-exonuclease, which removes the vast majority of 5’ mono-phosphorylated RNA from the sample (including 16S and 23S rRNA). b and c The XRN1 treated RNA is mixed with large excess of a synthetic RNA oligo (Rp6, shown in blue), and split into two pools. Both pools receive T4 RNA ligase, but only the “+RppH” pool is co-treated with RppH, an enzyme that converts 5’ tri-phosphorylated ends to mono-phosphorylated ends, thus allowing the ligase to use them as substrates. d and e After the ligation reaction, a semi-random primer is used to reverse-transcribe the RNA and simultaneously add a 2.0 Illumina adapter (black “B”). This results in cDNA with a 2.0 Illumina adaptor (for reverse reads in paired-end sequencing) at the 5’-end and if the template RNA was ligated to an Rp6 oligo, then the cDNA will also have a complementary sequence to Rp6 at the 3’-end (cRp6). f PCR is used to specifically amplify cDNAs that carry the 2.0 Illumina adaptor and cRp6 sequences. This step moreover adds a 1.0 Illumina adaptor (for forward reads in paired-end sequencing) and a sample-specific 4-base EMOTE barcode (blue line and “XXX”, respectively) to index the molecules (different barcodes for the -RppH and + RppH pools). The barcode of the -RppH pool will designate molecules where the XRN1 treatments has been incomplete, and this information is incorporated into the in silico analysis (see below). g The barcoded DNA from various samples (and pools) can be mixed, and loaded directly into an Illumina HiSeq machine. Millions of 50 nt sequences are obtained, each of which will span the EMOTE barcode, both known and random sections of the Rp6 oligo (see Methods ), and it will reveal the first 20 nt of the native 5’-end of the ligated RNA molecule. These 20 nt are sufficient to map the vast majority of 5’-ends to a unique position on the small genomes of the bacteria in this study. However, longer Illumina reads (and thus longer mapping sequences) can be used if the TSSs are in repeated regions or if large-genome organisms, such as humans, are being examined. h The in silico pipeline input consists of stranded RNA-seq reads for one or multiple biological replicates in FASTQ format. Each replicate includes a FASTQ for the -RppH pool and another for the + RppH pool. i The FASTQ files go through EMOTE-conv software [ 51 ] that parses the reads, aligns them to the genome, and perform the quantification. Thus, for each genomic position we obtain the number of reads whose first nucleotide align at this genomic position, and on which strand it maps. The counts are further corrected for PCR biases by looking at the unique molecular identifiers (UMIs) sequences available in the unaligned part of the EMOTE read. j Quantification counts obtained for + RppH and -RppH pools are compared through a beta-binomial model that tests whether the identified 5’ ends in the + RppH pool is significantly enriched over the identified 5’ ends in the -RppH pool at a given position. The process results in a p-value that reflects our confidence in the genomic position to be enriched in the + RppH pool of the biological replicate. k The p-values of all the biological replicates are combined into a single p-value with Fisher’s method. l and m To correct the p-values for multiple testing across all genomic positions, the false discovery rate (FDR) is evaluated and only those with a FDR ≤ 0.01 are considered to be TSSs. Note also that for the FDR is only calculated for genomic positions with at least 5 detected ligation-events in at least one of the + RppH pools (UMI ≥ 5). n The TSSs then enter an annotation process that retrieve their surrounding sequence and downstream ORFs. TSSs separated by less than 5 bp are clustered together. Finally, to draw a global picture of operon structures, an independent detection of transcription terminators is operated with the software TransTermHP [ 39 ]. o Sequence of the RNA oligo Rp6 and a typical Illumina sequencing read from a TSS-EMOTE experiment. The Recognition Sequence serves as priming site for the PCR in panel F. UMI: The randomly incorporated nucleotides in the Rp6 oligo that serves to whether Illumina reads with identical Mapping Sequences originate from separate ligation events. CS: Control Sequence. EB: EMOTE barcode to index the Illumina reads. An asterisk indicates the 5’ nucleotide of the original RNA molecule
    Figure Legend Snippet: TSS-EMOTE flowchart. The TSS-EMOTE assay consists of a wet-lab library preparation (panels a to g ) and in silico analyses (panel H to N). An asterisk continually marks the original 5’-base of tri-phosphorylated RNA (thin red line). a Total RNA is purified, and digested with XRN1 5’-exonuclease, which removes the vast majority of 5’ mono-phosphorylated RNA from the sample (including 16S and 23S rRNA). b and c The XRN1 treated RNA is mixed with large excess of a synthetic RNA oligo (Rp6, shown in blue), and split into two pools. Both pools receive T4 RNA ligase, but only the “+RppH” pool is co-treated with RppH, an enzyme that converts 5’ tri-phosphorylated ends to mono-phosphorylated ends, thus allowing the ligase to use them as substrates. d and e After the ligation reaction, a semi-random primer is used to reverse-transcribe the RNA and simultaneously add a 2.0 Illumina adapter (black “B”). This results in cDNA with a 2.0 Illumina adaptor (for reverse reads in paired-end sequencing) at the 5’-end and if the template RNA was ligated to an Rp6 oligo, then the cDNA will also have a complementary sequence to Rp6 at the 3’-end (cRp6). f PCR is used to specifically amplify cDNAs that carry the 2.0 Illumina adaptor and cRp6 sequences. This step moreover adds a 1.0 Illumina adaptor (for forward reads in paired-end sequencing) and a sample-specific 4-base EMOTE barcode (blue line and “XXX”, respectively) to index the molecules (different barcodes for the -RppH and + RppH pools). The barcode of the -RppH pool will designate molecules where the XRN1 treatments has been incomplete, and this information is incorporated into the in silico analysis (see below). g The barcoded DNA from various samples (and pools) can be mixed, and loaded directly into an Illumina HiSeq machine. Millions of 50 nt sequences are obtained, each of which will span the EMOTE barcode, both known and random sections of the Rp6 oligo (see Methods ), and it will reveal the first 20 nt of the native 5’-end of the ligated RNA molecule. These 20 nt are sufficient to map the vast majority of 5’-ends to a unique position on the small genomes of the bacteria in this study. However, longer Illumina reads (and thus longer mapping sequences) can be used if the TSSs are in repeated regions or if large-genome organisms, such as humans, are being examined. h The in silico pipeline input consists of stranded RNA-seq reads for one or multiple biological replicates in FASTQ format. Each replicate includes a FASTQ for the -RppH pool and another for the + RppH pool. i The FASTQ files go through EMOTE-conv software [ 51 ] that parses the reads, aligns them to the genome, and perform the quantification. Thus, for each genomic position we obtain the number of reads whose first nucleotide align at this genomic position, and on which strand it maps. The counts are further corrected for PCR biases by looking at the unique molecular identifiers (UMIs) sequences available in the unaligned part of the EMOTE read. j Quantification counts obtained for + RppH and -RppH pools are compared through a beta-binomial model that tests whether the identified 5’ ends in the + RppH pool is significantly enriched over the identified 5’ ends in the -RppH pool at a given position. The process results in a p-value that reflects our confidence in the genomic position to be enriched in the + RppH pool of the biological replicate. k The p-values of all the biological replicates are combined into a single p-value with Fisher’s method. l and m To correct the p-values for multiple testing across all genomic positions, the false discovery rate (FDR) is evaluated and only those with a FDR ≤ 0.01 are considered to be TSSs. Note also that for the FDR is only calculated for genomic positions with at least 5 detected ligation-events in at least one of the + RppH pools (UMI ≥ 5). n The TSSs then enter an annotation process that retrieve their surrounding sequence and downstream ORFs. TSSs separated by less than 5 bp are clustered together. Finally, to draw a global picture of operon structures, an independent detection of transcription terminators is operated with the software TransTermHP [ 39 ]. o Sequence of the RNA oligo Rp6 and a typical Illumina sequencing read from a TSS-EMOTE experiment. The Recognition Sequence serves as priming site for the PCR in panel F. UMI: The randomly incorporated nucleotides in the Rp6 oligo that serves to whether Illumina reads with identical Mapping Sequences originate from separate ligation events. CS: Control Sequence. EB: EMOTE barcode to index the Illumina reads. An asterisk indicates the 5’ nucleotide of the original RNA molecule

    Techniques Used: In Silico, Purification, Ligation, Sequencing, Polymerase Chain Reaction, RNA Sequencing Assay, Software

    5) Product Images from "Evolution of a transcriptional regulator from a transmembrane nucleoporin"

    Article Title: Evolution of a transcriptional regulator from a transmembrane nucleoporin

    Journal: Genes & Development

    doi: 10.1101/gad.280941.116

    Detection of sPom121 mRNA and protein expression in human cells. ( A ) Schematic of two putative Pom121 isoforms expressed in humans and confirmed by 5′ rapid amplification of cDNA ends (RACE). ( Top ) Gray boxes indicate 5′ UTR-encoding exons, blue boxes indicate TM domain-encoding exon, and black boxes indicate Pom121-encoding exons. ( Bottom ) The TM domain, nuclear localization signal (NLS), and phenylalanine/glycine (FG) domain are indicated (blue, red, and green, respectively). ( B ) Schematic showing the annotated 5′ end of the Pom121 gene (shown at the top , “Pom121 gene”) compared with that of sPom121 (“5′ RACE sPom121 mRNA”) and Pom121 (“5′ RACE Pom121 mRNA”), identified here by 5′ RACE. ( C ) Histone H3 Lys4 trimethylation (H3K4me3) ( top ) and RNA sequencing (RNA-seq) ( bottom ) results from HeLa-C cells. Red arrows are used to indicate active transcriptional start sites ( top ), sPom121-specific exons ( bottom left ), or the TM-coding exon of Pom121 ( bottom right ). ( D ) sPom121 and Pom121 expression in various tissues. Quantitative PCR (qPCR) analysis of sPom121 (red bars) and Pom121 (blue bars) mRNA levels in multiple tissue types relative to actin. Results are plotted such that the tissue with the lowest sPom121 mRNA expression is at the left , while the tissue expressing the highest levels of sPom121 is shown at the right . Different primers were used to analyze sPom121 and Pom121 cDNA levels, and thus a comparison of sPom121 and Pom121 levels in each tissue cannot be made from these data. ( E ) Western blot to detect sPom121. Soluble (lanes 1 , 2 ) and insoluble (lanes 3 , 4 ) lysates were electrophoresed and Western blotted, and proteins were detected with a Pom121 antibody ( top panels) or tubulin ( bottom panels). (Lanes 2 , 4 ) Samples that had been treated with Pom121 siRNA are included to identify which bands correspond to Pom121 protein. Pom121 blots were exposed for 30 sec ( left blot) or 10 sec ( right blot).
    Figure Legend Snippet: Detection of sPom121 mRNA and protein expression in human cells. ( A ) Schematic of two putative Pom121 isoforms expressed in humans and confirmed by 5′ rapid amplification of cDNA ends (RACE). ( Top ) Gray boxes indicate 5′ UTR-encoding exons, blue boxes indicate TM domain-encoding exon, and black boxes indicate Pom121-encoding exons. ( Bottom ) The TM domain, nuclear localization signal (NLS), and phenylalanine/glycine (FG) domain are indicated (blue, red, and green, respectively). ( B ) Schematic showing the annotated 5′ end of the Pom121 gene (shown at the top , “Pom121 gene”) compared with that of sPom121 (“5′ RACE sPom121 mRNA”) and Pom121 (“5′ RACE Pom121 mRNA”), identified here by 5′ RACE. ( C ) Histone H3 Lys4 trimethylation (H3K4me3) ( top ) and RNA sequencing (RNA-seq) ( bottom ) results from HeLa-C cells. Red arrows are used to indicate active transcriptional start sites ( top ), sPom121-specific exons ( bottom left ), or the TM-coding exon of Pom121 ( bottom right ). ( D ) sPom121 and Pom121 expression in various tissues. Quantitative PCR (qPCR) analysis of sPom121 (red bars) and Pom121 (blue bars) mRNA levels in multiple tissue types relative to actin. Results are plotted such that the tissue with the lowest sPom121 mRNA expression is at the left , while the tissue expressing the highest levels of sPom121 is shown at the right . Different primers were used to analyze sPom121 and Pom121 cDNA levels, and thus a comparison of sPom121 and Pom121 levels in each tissue cannot be made from these data. ( E ) Western blot to detect sPom121. Soluble (lanes 1 , 2 ) and insoluble (lanes 3 , 4 ) lysates were electrophoresed and Western blotted, and proteins were detected with a Pom121 antibody ( top panels) or tubulin ( bottom panels). (Lanes 2 , 4 ) Samples that had been treated with Pom121 siRNA are included to identify which bands correspond to Pom121 protein. Pom121 blots were exposed for 30 sec ( left blot) or 10 sec ( right blot).

    Techniques Used: Expressing, Rapid Amplification of cDNA Ends, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Western Blot, Size-exclusion Chromatography

    6) Product Images from "Chromatin-associated RNA sequencing (ChAR-seq) maps genome-wide RNA-to-DNA contacts"

    Article Title: Chromatin-associated RNA sequencing (ChAR-seq) maps genome-wide RNA-to-DNA contacts

    Journal: eLife

    doi: 10.7554/eLife.27024

    In vitro optimization of RNA-to-DNA ligation conditions. Upper panel , Ten pmols of 17-nt adenylated ssDNA oligonucleotide (Universal App DNA, CTGTAGGCACCATCAAT) was incubated with 5 pmols of a 17nt ssRNA test probe (TTTCGTTGGAAGCGGGA) in 1x NEB T4 RNA Ligase Buffer with the indicated ligase (NEB Thermostable 5’ AppDNA/RNA ligase (Therm 5' Ligase), NEB T4 Rnl2tr K227Q Ligase (trT4K) or NEB T4 Rnl2tr R55K, K227Q ligase (trT4KQ)) and/or supplements (PEG, BSA, ATP, RNaseOUT). Products were then analyzed using denaturing polyacrylamide gel electrophoresis using a combination of NEB microRNA and low range ssRNA ladders and stained with SYBR-gold. Bands were quantified and the percent product was calculated using (shifted / (total * 0.66)) to account for the molar excess of DNA over RNA. No adjustment was made to account for preferential staining of ssDNA over ssRNA. Residual signal is expected in the lower band owing to the molar excess of DNA over RNA. A high molecular weight band is visible in the Therm 5’ Ligase lane, which most likely consists of high molecular weight concatemers of the AppDNA substrate caused by incomplete 3’ blocking of these oligos or removal of the 3’ block by the Therm 5’ Ligase. This experiment was performed once.
    Figure Legend Snippet: In vitro optimization of RNA-to-DNA ligation conditions. Upper panel , Ten pmols of 17-nt adenylated ssDNA oligonucleotide (Universal App DNA, CTGTAGGCACCATCAAT) was incubated with 5 pmols of a 17nt ssRNA test probe (TTTCGTTGGAAGCGGGA) in 1x NEB T4 RNA Ligase Buffer with the indicated ligase (NEB Thermostable 5’ AppDNA/RNA ligase (Therm 5' Ligase), NEB T4 Rnl2tr K227Q Ligase (trT4K) or NEB T4 Rnl2tr R55K, K227Q ligase (trT4KQ)) and/or supplements (PEG, BSA, ATP, RNaseOUT). Products were then analyzed using denaturing polyacrylamide gel electrophoresis using a combination of NEB microRNA and low range ssRNA ladders and stained with SYBR-gold. Bands were quantified and the percent product was calculated using (shifted / (total * 0.66)) to account for the molar excess of DNA over RNA. No adjustment was made to account for preferential staining of ssDNA over ssRNA. Residual signal is expected in the lower band owing to the molar excess of DNA over RNA. A high molecular weight band is visible in the Therm 5’ Ligase lane, which most likely consists of high molecular weight concatemers of the AppDNA substrate caused by incomplete 3’ blocking of these oligos or removal of the 3’ block by the Therm 5’ Ligase. This experiment was performed once.

    Techniques Used: In Vitro, DNA Ligation, Incubation, Polyacrylamide Gel Electrophoresis, Staining, Molecular Weight, Blocking Assay

    7) Product Images from "TSS-EMOTE, a refined protocol for a more complete and less biased global mapping of transcription start sites in bacterial pathogens"

    Article Title: TSS-EMOTE, a refined protocol for a more complete and less biased global mapping of transcription start sites in bacterial pathogens

    Journal: BMC Genomics

    doi: 10.1186/s12864-016-3211-3

    TSS-EMOTE flowchart. The TSS-EMOTE assay consists of a wet-lab library preparation (panels a to g ) and in silico analyses (panel H to N). An asterisk continually marks the original 5’-base of tri-phosphorylated RNA (thin red line). a Total RNA is purified, and digested with XRN1 5’-exonuclease, which removes the vast majority of 5’ mono-phosphorylated RNA from the sample (including 16S and 23S rRNA). b and c The XRN1 treated RNA is mixed with large excess of a synthetic RNA oligo (Rp6, shown in blue), and split into two pools. Both pools receive T4 RNA ligase, but only the “+RppH” pool is co-treated with RppH, an enzyme that converts 5’ tri-phosphorylated ends to mono-phosphorylated ends, thus allowing the ligase to use them as substrates. d and e After the ligation reaction, a semi-random primer is used to reverse-transcribe the RNA and simultaneously add a 2.0 Illumina adapter (black “B”). This results in cDNA with a 2.0 Illumina adaptor (for reverse reads in paired-end sequencing) at the 5’-end and if the template RNA was ligated to an Rp6 oligo, then the cDNA will also have a complementary sequence to Rp6 at the 3’-end (cRp6). f PCR is used to specifically amplify cDNAs that carry the 2.0 Illumina adaptor and cRp6 sequences. This step moreover adds a 1.0 Illumina adaptor (for forward reads in paired-end sequencing) and a sample-specific 4-base EMOTE barcode (blue line and “XXX”, respectively) to index the molecules (different barcodes for the -RppH and + RppH pools). The barcode of the -RppH pool will designate molecules where the XRN1 treatments has been incomplete, and this information is incorporated into the in silico analysis (see below). g The barcoded DNA from various samples (and pools) can be mixed, and loaded directly into an Illumina HiSeq machine. Millions of 50 nt sequences are obtained, each of which will span the EMOTE barcode, both known and random sections of the Rp6 oligo (see Methods ), and it will reveal the first 20 nt of the native 5’-end of the ligated RNA molecule. These 20 nt are sufficient to map the vast majority of 5’-ends to a unique position on the small genomes of the bacteria in this study. However, longer Illumina reads (and thus longer mapping sequences) can be used if the TSSs are in repeated regions or if large-genome organisms, such as humans, are being examined. h The in silico pipeline input consists of stranded RNA-seq reads for one or multiple biological replicates in FASTQ format. Each replicate includes a FASTQ for the -RppH pool and another for the + RppH pool. i The FASTQ files go through EMOTE-conv software [ 51 ] that parses the reads, aligns them to the genome, and perform the quantification. Thus, for each genomic position we obtain the number of reads whose first nucleotide align at this genomic position, and on which strand it maps. The counts are further corrected for PCR biases by looking at the unique molecular identifiers (UMIs) sequences available in the unaligned part of the EMOTE read. j Quantification counts obtained for + RppH and -RppH pools are compared through a beta-binomial model that tests whether the identified 5’ ends in the + RppH pool is significantly enriched over the identified 5’ ends in the -RppH pool at a given position. The process results in a p-value that reflects our confidence in the genomic position to be enriched in the + RppH pool of the biological replicate. k The p-values of all the biological replicates are combined into a single p-value with Fisher’s method. l and m To correct the p-values for multiple testing across all genomic positions, the false discovery rate (FDR) is evaluated and only those with a FDR ≤ 0.01 are considered to be TSSs. Note also that for the FDR is only calculated for genomic positions with at least 5 detected ligation-events in at least one of the + RppH pools (UMI ≥ 5). n The TSSs then enter an annotation process that retrieve their surrounding sequence and downstream ORFs. TSSs separated by less than 5 bp are clustered together. Finally, to draw a global picture of operon structures, an independent detection of transcription terminators is operated with the software TransTermHP [ 39 ]. o Sequence of the RNA oligo Rp6 and a typical Illumina sequencing read from a TSS-EMOTE experiment. The Recognition Sequence serves as priming site for the PCR in panel F. UMI: The randomly incorporated nucleotides in the Rp6 oligo that serves to whether Illumina reads with identical Mapping Sequences originate from separate ligation events. CS: Control Sequence. EB: EMOTE barcode to index the Illumina reads. An asterisk indicates the 5’ nucleotide of the original RNA molecule
    Figure Legend Snippet: TSS-EMOTE flowchart. The TSS-EMOTE assay consists of a wet-lab library preparation (panels a to g ) and in silico analyses (panel H to N). An asterisk continually marks the original 5’-base of tri-phosphorylated RNA (thin red line). a Total RNA is purified, and digested with XRN1 5’-exonuclease, which removes the vast majority of 5’ mono-phosphorylated RNA from the sample (including 16S and 23S rRNA). b and c The XRN1 treated RNA is mixed with large excess of a synthetic RNA oligo (Rp6, shown in blue), and split into two pools. Both pools receive T4 RNA ligase, but only the “+RppH” pool is co-treated with RppH, an enzyme that converts 5’ tri-phosphorylated ends to mono-phosphorylated ends, thus allowing the ligase to use them as substrates. d and e After the ligation reaction, a semi-random primer is used to reverse-transcribe the RNA and simultaneously add a 2.0 Illumina adapter (black “B”). This results in cDNA with a 2.0 Illumina adaptor (for reverse reads in paired-end sequencing) at the 5’-end and if the template RNA was ligated to an Rp6 oligo, then the cDNA will also have a complementary sequence to Rp6 at the 3’-end (cRp6). f PCR is used to specifically amplify cDNAs that carry the 2.0 Illumina adaptor and cRp6 sequences. This step moreover adds a 1.0 Illumina adaptor (for forward reads in paired-end sequencing) and a sample-specific 4-base EMOTE barcode (blue line and “XXX”, respectively) to index the molecules (different barcodes for the -RppH and + RppH pools). The barcode of the -RppH pool will designate molecules where the XRN1 treatments has been incomplete, and this information is incorporated into the in silico analysis (see below). g The barcoded DNA from various samples (and pools) can be mixed, and loaded directly into an Illumina HiSeq machine. Millions of 50 nt sequences are obtained, each of which will span the EMOTE barcode, both known and random sections of the Rp6 oligo (see Methods ), and it will reveal the first 20 nt of the native 5’-end of the ligated RNA molecule. These 20 nt are sufficient to map the vast majority of 5’-ends to a unique position on the small genomes of the bacteria in this study. However, longer Illumina reads (and thus longer mapping sequences) can be used if the TSSs are in repeated regions or if large-genome organisms, such as humans, are being examined. h The in silico pipeline input consists of stranded RNA-seq reads for one or multiple biological replicates in FASTQ format. Each replicate includes a FASTQ for the -RppH pool and another for the + RppH pool. i The FASTQ files go through EMOTE-conv software [ 51 ] that parses the reads, aligns them to the genome, and perform the quantification. Thus, for each genomic position we obtain the number of reads whose first nucleotide align at this genomic position, and on which strand it maps. The counts are further corrected for PCR biases by looking at the unique molecular identifiers (UMIs) sequences available in the unaligned part of the EMOTE read. j Quantification counts obtained for + RppH and -RppH pools are compared through a beta-binomial model that tests whether the identified 5’ ends in the + RppH pool is significantly enriched over the identified 5’ ends in the -RppH pool at a given position. The process results in a p-value that reflects our confidence in the genomic position to be enriched in the + RppH pool of the biological replicate. k The p-values of all the biological replicates are combined into a single p-value with Fisher’s method. l and m To correct the p-values for multiple testing across all genomic positions, the false discovery rate (FDR) is evaluated and only those with a FDR ≤ 0.01 are considered to be TSSs. Note also that for the FDR is only calculated for genomic positions with at least 5 detected ligation-events in at least one of the + RppH pools (UMI ≥ 5). n The TSSs then enter an annotation process that retrieve their surrounding sequence and downstream ORFs. TSSs separated by less than 5 bp are clustered together. Finally, to draw a global picture of operon structures, an independent detection of transcription terminators is operated with the software TransTermHP [ 39 ]. o Sequence of the RNA oligo Rp6 and a typical Illumina sequencing read from a TSS-EMOTE experiment. The Recognition Sequence serves as priming site for the PCR in panel F. UMI: The randomly incorporated nucleotides in the Rp6 oligo that serves to whether Illumina reads with identical Mapping Sequences originate from separate ligation events. CS: Control Sequence. EB: EMOTE barcode to index the Illumina reads. An asterisk indicates the 5’ nucleotide of the original RNA molecule

    Techniques Used: In Silico, Purification, Ligation, Sequencing, Polymerase Chain Reaction, RNA Sequencing Assay, Software

    8) Product Images from "SCRINSHOT, a spatial method for single-cell resolution mapping of cell states in tissue sections"

    Article Title: SCRINSHOT, a spatial method for single-cell resolution mapping of cell states in tissue sections

    Journal: bioRxiv

    doi: 10.1101/2020.02.07.938571

    SCRINSHOT specificity relies on stringent hybridization of padlock probes to their target RNAs. Images of SCRINSHOT signal, using normal Scgb1a1 padlock probe (A), a Scgb1a1 padlock probe with a point mutation at its ligation site (B), a Scgb1a1 padlock probe with 3’-scrambled arm (C) and normal padlock probe but omitting SplintR ligase (D). Actb normal padlock probe was used in all conditions as internal control. DAPI: blue, Scgb1a1 : gray, Actb : red. “n” indicates the number of airway cells in the corresponding images. (A’-D’) Magnified areas of the indicated positions (square brackets) of images in the left. Pink outlines show the 2 μm expanded airway nuclear ROIs, which are considered as cells. Scale-bar: 150 μm. (E) Violin plot of the Scgb1a1 and Actb signal-dots ratio in all airway cells. The ratio of cells with zero Actb -dots considered as zero. (F) Violin plot of the Scgb1a1 and Actb fluorescence intensity ratio in all airway cells. SplintR pos n=473, mismatch n=574, 3’-scrambled n=507 and SplintR neg n=488.
    Figure Legend Snippet: SCRINSHOT specificity relies on stringent hybridization of padlock probes to their target RNAs. Images of SCRINSHOT signal, using normal Scgb1a1 padlock probe (A), a Scgb1a1 padlock probe with a point mutation at its ligation site (B), a Scgb1a1 padlock probe with 3’-scrambled arm (C) and normal padlock probe but omitting SplintR ligase (D). Actb normal padlock probe was used in all conditions as internal control. DAPI: blue, Scgb1a1 : gray, Actb : red. “n” indicates the number of airway cells in the corresponding images. (A’-D’) Magnified areas of the indicated positions (square brackets) of images in the left. Pink outlines show the 2 μm expanded airway nuclear ROIs, which are considered as cells. Scale-bar: 150 μm. (E) Violin plot of the Scgb1a1 and Actb signal-dots ratio in all airway cells. The ratio of cells with zero Actb -dots considered as zero. (F) Violin plot of the Scgb1a1 and Actb fluorescence intensity ratio in all airway cells. SplintR pos n=473, mismatch n=574, 3’-scrambled n=507 and SplintR neg n=488.

    Techniques Used: Hybridization, Mutagenesis, Ligation, Fluorescence

    Comparison of SplintR-based (SCRINSHOT) and the cDNA-based in situ hybridization assays for high, intermediate and low abundant genes in sequential PFA fixed lung sections. (A) Images of SplintR-based (SCRINSHOT) and cDNA-based in situ hybridization assays, in sequential lung sections. DAPI: blue, Scgb1a1 : green, Sftpc : gray, Actb : red and Pecam1 : cyan. Pink outlines show the 2 μm expanded airway nuclear ROIs, which are considered as cells. The square brackets indicate the magnified areas on the right. The “n” correspond to the number of counted cells in large images. Scale bar: 100μm. (B) Bar-plots of the analyzed gene signals, in the indicated tissue compartments, for SCRINSHOT and cDNA-based approaches. The differences between the two conditions are significant (P
    Figure Legend Snippet: Comparison of SplintR-based (SCRINSHOT) and the cDNA-based in situ hybridization assays for high, intermediate and low abundant genes in sequential PFA fixed lung sections. (A) Images of SplintR-based (SCRINSHOT) and cDNA-based in situ hybridization assays, in sequential lung sections. DAPI: blue, Scgb1a1 : green, Sftpc : gray, Actb : red and Pecam1 : cyan. Pink outlines show the 2 μm expanded airway nuclear ROIs, which are considered as cells. The square brackets indicate the magnified areas on the right. The “n” correspond to the number of counted cells in large images. Scale bar: 100μm. (B) Bar-plots of the analyzed gene signals, in the indicated tissue compartments, for SCRINSHOT and cDNA-based approaches. The differences between the two conditions are significant (P

    Techniques Used: In Situ Hybridization

    9) Product Images from "Four Methods of Preparing mRNA 5? End Libraries Using the Illumina Sequencing Platform"

    Article Title: Four Methods of Preparing mRNA 5? End Libraries Using the Illumina Sequencing Platform

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0101812

    Library preparation using the CapSMART method. A) The protocol used either poly A+ (0.50–10 µg) or total (10–200 µg) RNA. B) De-phosphorylation of mono-, di-, and tri- phosphate groups from non-capped 5′ end molecules using alkaline phosphatase. C) Phosphorylation to add mono-phosphate to the non-capped 5′ end molecules using T4 Polynucleotide Kinase. D) Ligation of STOP oligos. A total of three kinds of oligonucleotides ( Table 2 : STOP1: iGiCiG, STOP2: iCiGiC, STOPMix: mixture of STOP1 and STOP2) were used in the present study. E) First-strand cDNA synthesis. F) Second-strand cDNA amplification by PCR with biotinylated 5′ end primers. G) Fragmentation of cDNA using a Bioruptor and collection of biotinylated 5′ ends using beads. H) Illumina sequencing library preparation.
    Figure Legend Snippet: Library preparation using the CapSMART method. A) The protocol used either poly A+ (0.50–10 µg) or total (10–200 µg) RNA. B) De-phosphorylation of mono-, di-, and tri- phosphate groups from non-capped 5′ end molecules using alkaline phosphatase. C) Phosphorylation to add mono-phosphate to the non-capped 5′ end molecules using T4 Polynucleotide Kinase. D) Ligation of STOP oligos. A total of three kinds of oligonucleotides ( Table 2 : STOP1: iGiCiG, STOP2: iCiGiC, STOPMix: mixture of STOP1 and STOP2) were used in the present study. E) First-strand cDNA synthesis. F) Second-strand cDNA amplification by PCR with biotinylated 5′ end primers. G) Fragmentation of cDNA using a Bioruptor and collection of biotinylated 5′ ends using beads. H) Illumina sequencing library preparation.

    Techniques Used: De-Phosphorylation Assay, Ligation, Amplification, Polymerase Chain Reaction, Sequencing

    10) Product Images from "3′ Branch ligation: a novel method to ligate non-complementary DNA to recessed or internal 3′OH ends in DNA or RNA"

    Article Title: 3′ Branch ligation: a novel method to ligate non-complementary DNA to recessed or internal 3′OH ends in DNA or RNA

    Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

    doi: 10.1093/dnares/dsy037

    3′ Branch ligation at the 3′ end of RNA in DNA/RNA hybrid. Schematic representation of 3′-branch ligation on a DNA/RNA hybrid with a 20‐bp complimentary region. We tested whether blunt-end DNA donors would ligate to the 3 ′ -recessive end of DNA and/or to the 3 ′ -recessive end of RNA. DNA(ON-21) hybridizes with the RNA strand (a), whereas DNA(ON-23) cannot hybridize with the RNA strand (b). (c, d) Gel analysis of size shift of ligated products using 6% denaturing polyacrylamide gel. The red arrowheads correspond to the RNA substrate (29 nt), and the green arrowhead corresponds to DNA substrate (80 nt). The purple arrowhead corresponds to donor-ligated RNA substrates. If ligation occurs, the substrate size would shift up by 20 nt. (c) Lanes 1 and 2, experimental duplicates; lanes 7–10, no-ligase controls; 10% PEG was added with T4 DNA ligase. (d) Lane 1, no-ligase control; lanes 2, 3, and 8, T4 DNA ligase with 10% PEG; lanes 4, 5, and 9, T4 RNA ligase 1 with 20% DMSO; lanes 6, 7, and 10, T4 RNA ligase 2 with 20% DMSO.
    Figure Legend Snippet: 3′ Branch ligation at the 3′ end of RNA in DNA/RNA hybrid. Schematic representation of 3′-branch ligation on a DNA/RNA hybrid with a 20‐bp complimentary region. We tested whether blunt-end DNA donors would ligate to the 3 ′ -recessive end of DNA and/or to the 3 ′ -recessive end of RNA. DNA(ON-21) hybridizes with the RNA strand (a), whereas DNA(ON-23) cannot hybridize with the RNA strand (b). (c, d) Gel analysis of size shift of ligated products using 6% denaturing polyacrylamide gel. The red arrowheads correspond to the RNA substrate (29 nt), and the green arrowhead corresponds to DNA substrate (80 nt). The purple arrowhead corresponds to donor-ligated RNA substrates. If ligation occurs, the substrate size would shift up by 20 nt. (c) Lanes 1 and 2, experimental duplicates; lanes 7–10, no-ligase controls; 10% PEG was added with T4 DNA ligase. (d) Lane 1, no-ligase control; lanes 2, 3, and 8, T4 DNA ligase with 10% PEG; lanes 4, 5, and 9, T4 RNA ligase 1 with 20% DMSO; lanes 6, 7, and 10, T4 RNA ligase 2 with 20% DMSO.

    Techniques Used: Ligation

    11) Product Images from "Elimination of Ligation Dependent Artifacts in T4 RNA Ligase to Achieve High Efficiency and Low Bias MicroRNA Capture"

    Article Title: Elimination of Ligation Dependent Artifacts in T4 RNA Ligase to Achieve High Efficiency and Low Bias MicroRNA Capture

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0094619

    MicroRNA capture was performed with 4 different ligases using the vendor recommended protocols to compare capture efficiency across 20 different microRNA. The ligation products were analyzed by 15% denaturing urea-PAGE. Capture efficiency was determined by performing a Cy3 scan and comparing the intensities of the ∼40 nt captured microRNA band versus the ∼20 nt free microRNA band. T4 RNA Ligase 2 truncated (T4 Rnl2 T) had high average capture efficiency and low bias but many randomly sized background products. The point mutant enzymes T4 RNA Ligase 2 truncated K227Q (T4 Rnl2 TK) and T4 RNA Ligase 2 truncated KQ (T4 Rnl2 TKQ) had decreased side product formation but also lower average capture efficiency and higher bias. Thermostable 5′ App DNA/RNA Ligase (Mth Rnl), which was performed at 65°C instead of 25°C, had similar average capture efficiency and bias but with distinct ligation efficiency pattern.
    Figure Legend Snippet: MicroRNA capture was performed with 4 different ligases using the vendor recommended protocols to compare capture efficiency across 20 different microRNA. The ligation products were analyzed by 15% denaturing urea-PAGE. Capture efficiency was determined by performing a Cy3 scan and comparing the intensities of the ∼40 nt captured microRNA band versus the ∼20 nt free microRNA band. T4 RNA Ligase 2 truncated (T4 Rnl2 T) had high average capture efficiency and low bias but many randomly sized background products. The point mutant enzymes T4 RNA Ligase 2 truncated K227Q (T4 Rnl2 TK) and T4 RNA Ligase 2 truncated KQ (T4 Rnl2 TKQ) had decreased side product formation but also lower average capture efficiency and higher bias. Thermostable 5′ App DNA/RNA Ligase (Mth Rnl), which was performed at 65°C instead of 25°C, had similar average capture efficiency and bias but with distinct ligation efficiency pattern.

    Techniques Used: Ligation, Polyacrylamide Gel Electrophoresis, Mutagenesis

    Schematic illustration of microRNA capture by 3′ adapter ligation. The 19 nt, enzymatically pre-adenlyated adapter is ligated to the 3′ OH of microRNA using T4 RNA ligase 2. The reaction is run at 25°C for 4 hours in the absence of ATP. In order to characterize capture efficiency, the microRNA is end labeled with Cy3. The 3′ end of the adapter is blocked by –ddC, a fluorophore, or other moiety to prevent the formation of concatemers and circularized products.
    Figure Legend Snippet: Schematic illustration of microRNA capture by 3′ adapter ligation. The 19 nt, enzymatically pre-adenlyated adapter is ligated to the 3′ OH of microRNA using T4 RNA ligase 2. The reaction is run at 25°C for 4 hours in the absence of ATP. In order to characterize capture efficiency, the microRNA is end labeled with Cy3. The 3′ end of the adapter is blocked by –ddC, a fluorophore, or other moiety to prevent the formation of concatemers and circularized products.

    Techniques Used: Ligation, Labeling

    12) Product Images from "SCRINSHOT, a spatial method for single-cell resolution mapping of cell states in tissue sections"

    Article Title: SCRINSHOT, a spatial method for single-cell resolution mapping of cell states in tissue sections

    Journal: bioRxiv

    doi: 10.1101/2020.02.07.938571

    SCRINSHOT specificity relies on stringent hybridization of padlock probes to their target RNAs. Images of SCRINSHOT signal, using normal Scgb1a1 padlock probe (A), a Scgb1a1 padlock probe with a point mutation at its ligation site (B), a Scgb1a1 padlock probe with 3’-scrambled arm (C) and normal padlock probe but omitting SplintR ligase (D). Actb normal padlock probe was used in all conditions as internal control. DAPI: blue, Scgb1a1 : gray, Actb : red. “n” indicates the number of airway cells in the corresponding images. (A’-D’) Magnified areas of the indicated positions (square brackets) of images in the left. Pink outlines show the 2 μm expanded airway nuclear ROIs, which are considered as cells. Scale-bar: 150 μm. (E) Violin plot of the Scgb1a1 and Actb signal-dots ratio in all airway cells. The ratio of cells with zero Actb -dots considered as zero. (F) Violin plot of the Scgb1a1 and Actb fluorescence intensity ratio in all airway cells. SplintR pos n=473, mismatch n=574, 3’-scrambled n=507 and SplintR neg n=488.
    Figure Legend Snippet: SCRINSHOT specificity relies on stringent hybridization of padlock probes to their target RNAs. Images of SCRINSHOT signal, using normal Scgb1a1 padlock probe (A), a Scgb1a1 padlock probe with a point mutation at its ligation site (B), a Scgb1a1 padlock probe with 3’-scrambled arm (C) and normal padlock probe but omitting SplintR ligase (D). Actb normal padlock probe was used in all conditions as internal control. DAPI: blue, Scgb1a1 : gray, Actb : red. “n” indicates the number of airway cells in the corresponding images. (A’-D’) Magnified areas of the indicated positions (square brackets) of images in the left. Pink outlines show the 2 μm expanded airway nuclear ROIs, which are considered as cells. Scale-bar: 150 μm. (E) Violin plot of the Scgb1a1 and Actb signal-dots ratio in all airway cells. The ratio of cells with zero Actb -dots considered as zero. (F) Violin plot of the Scgb1a1 and Actb fluorescence intensity ratio in all airway cells. SplintR pos n=473, mismatch n=574, 3’-scrambled n=507 and SplintR neg n=488.

    Techniques Used: Hybridization, Mutagenesis, Ligation, Fluorescence

    Comparison of SplintR-based (SCRINSHOT) and the cDNA-based in situ hybridization assays for high, intermediate and low abundant genes in sequential PFA fixed lung sections. (A) Images of SplintR-based (SCRINSHOT) and cDNA-based in situ hybridization assays, in sequential lung sections. DAPI: blue, Scgb1a1 : green, Sftpc : gray, Actb : red and Pecam1 : cyan. Pink outlines show the 2 μm expanded airway nuclear ROIs, which are considered as cells. The square brackets indicate the magnified areas on the right. The “n” correspond to the number of counted cells in large images. Scale bar: 100μm. (B) Bar-plots of the analyzed gene signals, in the indicated tissue compartments, for SCRINSHOT and cDNA-based approaches. The differences between the two conditions are significant (P
    Figure Legend Snippet: Comparison of SplintR-based (SCRINSHOT) and the cDNA-based in situ hybridization assays for high, intermediate and low abundant genes in sequential PFA fixed lung sections. (A) Images of SplintR-based (SCRINSHOT) and cDNA-based in situ hybridization assays, in sequential lung sections. DAPI: blue, Scgb1a1 : green, Sftpc : gray, Actb : red and Pecam1 : cyan. Pink outlines show the 2 μm expanded airway nuclear ROIs, which are considered as cells. The square brackets indicate the magnified areas on the right. The “n” correspond to the number of counted cells in large images. Scale bar: 100μm. (B) Bar-plots of the analyzed gene signals, in the indicated tissue compartments, for SCRINSHOT and cDNA-based approaches. The differences between the two conditions are significant (P

    Techniques Used: In Situ Hybridization

    Related Articles

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    Article Title: Loss of a Universal tRNA Feature ▿
    Article Snippet: .. For rephosphorylation, dephosphorylated RNA was incubated at 125 ng/μl for 50 min at 37°C in RNA ligase buffer with 0.5 U of T4 polynucleotide kinase (New England Biolabs)/μl, purifying the product RNA as described above. .. Reverse transcription-PCR (RT-PCR) primers (-R and -F) are indicated in Fig. .

    Article Title: Late steps of ribosome assembly in E. coli are sensitive to a severe heat stress but are assisted by the HSP70 chaperone machine †
    Article Snippet: .. Thermo-denaturation of the RNA prior to the 3′5′ RACE Five micrograms of RNA in a total volume of 20 µl containing the T4 RNA ligase buffer (New England Biolabs, n° B0204S) were incubated for 4 min at 75°C, and then rapidly frozen in a solid CO2 (dry ice)-ethanol bath for 1 min. To allow the samples to thaw slowly, they were then placed on ice for about 15 min, as described in ref. 22, and then 2 µl of T4 RNA ligase was added to start the 3′5′ RNA ligation. ..

    Article Title: Strand-specific deep sequencing of the transcriptome
    Article Snippet: .. We incubated the following reaction mixture for 30 min at 37°C: 10 μL of sample, 1 μL of 10× T4 RNA ligase buffer (as fresh ATP supply), 10 U of polynucleotide kinase (New England BioLabs), 3 μL of RNase free water. .. After addition of 2× loading dye and incubation for 5 min at 65°C, the reaction was loaded onto a denaturing urea-PAGE gel in order to separate the fragments with ligated 3′ adapter from nonligated adapter (band sizes: insert size + 23 nt for single end sequencing; insert size + 34 nt for paired end libraries).

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    Article Title: Chromatin-associated RNA sequencing (ChAR-seq) maps genome-wide RNA-to-DNA contacts
    Article Snippet: Step 4B ( Optional additional step B ): RNase control Pre-mix and resuspend the pellet in the following 160 µL water 20 µL 10x T4 RNA ligase buffer 10 µL 10 mg/mL RNaseA 10 µL RNaseH (NEB) Incubate at 37C for 4 hr Centrifuge at 2.5 k x g for 2 min, discard supernatant Add 1000 µL DEPC-treated PBS, mix gently Centrifuge at 2.5 k x g for 2 min, discard supernatant Immediately proceed to the next step, with pre-mixed reaction buffer already prepared

    Ligation:

    Article Title: Late steps of ribosome assembly in E. coli are sensitive to a severe heat stress but are assisted by the HSP70 chaperone machine †
    Article Snippet: .. Thermo-denaturation of the RNA prior to the 3′5′ RACE Five micrograms of RNA in a total volume of 20 µl containing the T4 RNA ligase buffer (New England Biolabs, n° B0204S) were incubated for 4 min at 75°C, and then rapidly frozen in a solid CO2 (dry ice)-ethanol bath for 1 min. To allow the samples to thaw slowly, they were then placed on ice for about 15 min, as described in ref. 22, and then 2 µl of T4 RNA ligase was added to start the 3′5′ RNA ligation. ..

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    New England Biolabs t4 rna ligase buffer
    DSSS protocol workflow. ( A ) Fragmentation. RNA is fragmented to sizes in the range of 60–200 nt. ( B ) Dephosphorylation. 5′ phosphates are removed from RNA by treatment with alkaline phosphatase. ( C ) 3′ adapter ligation. Dephosphorylated 200-nt-long RNA fragments are selected by urea-PAGE. The 3′ adapter is ligated to the 3′ ends using <t>T4</t> RNA ligase I. ( D ) Rephosphorylation. Fragments are rephosphorylated by treatment with T4 polynucleotide kinase as preparation for the next ligation step. ( E ) 5′ adapter ligation, preceded by removal of the nonligated 3′adapter by urea-PAGE size selection. ( F ) Reverse transcription (RT) and amplification of library. Molecules with 5′ and 3′ adapters were selected by urea-PAGE. First strand cDNA synthesis and PCR amplification were carried out with the indicated primers. ( G ) Sequencing.
    T4 Rna Ligase Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    DSSS protocol workflow. ( A ) Fragmentation. RNA is fragmented to sizes in the range of 60–200 nt. ( B ) Dephosphorylation. 5′ phosphates are removed from RNA by treatment with alkaline phosphatase. ( C ) 3′ adapter ligation. Dephosphorylated 200-nt-long RNA fragments are selected by urea-PAGE. The 3′ adapter is ligated to the 3′ ends using T4 RNA ligase I. ( D ) Rephosphorylation. Fragments are rephosphorylated by treatment with T4 polynucleotide kinase as preparation for the next ligation step. ( E ) 5′ adapter ligation, preceded by removal of the nonligated 3′adapter by urea-PAGE size selection. ( F ) Reverse transcription (RT) and amplification of library. Molecules with 5′ and 3′ adapters were selected by urea-PAGE. First strand cDNA synthesis and PCR amplification were carried out with the indicated primers. ( G ) Sequencing.

    Journal: Genome Research

    Article Title: Strand-specific deep sequencing of the transcriptome

    doi: 10.1101/gr.094318.109

    Figure Lengend Snippet: DSSS protocol workflow. ( A ) Fragmentation. RNA is fragmented to sizes in the range of 60–200 nt. ( B ) Dephosphorylation. 5′ phosphates are removed from RNA by treatment with alkaline phosphatase. ( C ) 3′ adapter ligation. Dephosphorylated 200-nt-long RNA fragments are selected by urea-PAGE. The 3′ adapter is ligated to the 3′ ends using T4 RNA ligase I. ( D ) Rephosphorylation. Fragments are rephosphorylated by treatment with T4 polynucleotide kinase as preparation for the next ligation step. ( E ) 5′ adapter ligation, preceded by removal of the nonligated 3′adapter by urea-PAGE size selection. ( F ) Reverse transcription (RT) and amplification of library. Molecules with 5′ and 3′ adapters were selected by urea-PAGE. First strand cDNA synthesis and PCR amplification were carried out with the indicated primers. ( G ) Sequencing.

    Article Snippet: We incubated the following reaction mixture for 30 min at 37°C: 10 μL of sample, 1 μL of 10× T4 RNA ligase buffer (as fresh ATP supply), 10 U of polynucleotide kinase (New England BioLabs), 3 μL of RNase free water.

    Techniques: De-Phosphorylation Assay, Ligation, Polyacrylamide Gel Electrophoresis, Selection, Amplification, Polymerase Chain Reaction, Sequencing

    ( A ) Schematic processing of the p1 16S rRNA. The extra-sequences of 115 nt and 33 nt, flanking the m 16S rRNA at its 5′ and 3′ ends, respectively, are shown on a grey background. RA and FA are the primers used for 3′5′ RACE analysis. The site of annealing of RA to m 16S rRNA, and that of FA to the reverse complement of the m 16S rRNA, are indicated by arrows. Figure not drawn to scale. ( B ) Expected sizes in bp of the RT-PCR products (amplicons) obtained from the different species of 16S rRNA ( p1 , p2 , p3 and m ) by 3′5′ RACE. ( C–F ) Agarose gel electrophoresis of RT-PCR products obtained by 3′5′ RACE from total RNA isolated from MC4100 bacteria grown at 30°C (C), or 44°C (D), or 45°C (E) or 46°C (F). Each RNA sample was thermo-denatured (lanes b), or not (lanes a) prior to the 3′5′ ligation. The sizes (in bp) of the molecular weight markers are indicated to the left of each gel (M). ( G ) The thermodenaturation step dissociates the complementary sequences present at the 3′ and 5′ends of the p1 16S rRNA, and therefore offers to all the 16S rRNA species an equal chance to access to the T4 RNA ligase.

    Journal: Nucleic Acids Research

    Article Title: Late steps of ribosome assembly in E. coli are sensitive to a severe heat stress but are assisted by the HSP70 chaperone machine †

    doi: 10.1093/nar/gkq1049

    Figure Lengend Snippet: ( A ) Schematic processing of the p1 16S rRNA. The extra-sequences of 115 nt and 33 nt, flanking the m 16S rRNA at its 5′ and 3′ ends, respectively, are shown on a grey background. RA and FA are the primers used for 3′5′ RACE analysis. The site of annealing of RA to m 16S rRNA, and that of FA to the reverse complement of the m 16S rRNA, are indicated by arrows. Figure not drawn to scale. ( B ) Expected sizes in bp of the RT-PCR products (amplicons) obtained from the different species of 16S rRNA ( p1 , p2 , p3 and m ) by 3′5′ RACE. ( C–F ) Agarose gel electrophoresis of RT-PCR products obtained by 3′5′ RACE from total RNA isolated from MC4100 bacteria grown at 30°C (C), or 44°C (D), or 45°C (E) or 46°C (F). Each RNA sample was thermo-denatured (lanes b), or not (lanes a) prior to the 3′5′ ligation. The sizes (in bp) of the molecular weight markers are indicated to the left of each gel (M). ( G ) The thermodenaturation step dissociates the complementary sequences present at the 3′ and 5′ends of the p1 16S rRNA, and therefore offers to all the 16S rRNA species an equal chance to access to the T4 RNA ligase.

    Article Snippet: Thermo-denaturation of the RNA prior to the 3′5′ RACE Five micrograms of RNA in a total volume of 20 µl containing the T4 RNA ligase buffer (New England Biolabs, n° B0204S) were incubated for 4 min at 75°C, and then rapidly frozen in a solid CO2 (dry ice)-ethanol bath for 1 min. To allow the samples to thaw slowly, they were then placed on ice for about 15 min, as described in ref. 22, and then 2 µl of T4 RNA ligase was added to start the 3′5′ RNA ligation.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Isolation, Ligation, Molecular Weight

    tRNA end sequence analysis. A-C. RNA ligation products. Joints formed by T4 RNA ligase (brackets), involving the circled 5′-monophosphate ends, as revealed by RT-PCR with the indicated primers. Sequences of precursor RNA sequences are shown with

    Journal:

    Article Title: Loss of a Universal tRNA Feature ▿

    doi: 10.1128/JB.01203-06

    Figure Lengend Snippet: tRNA end sequence analysis. A-C. RNA ligation products. Joints formed by T4 RNA ligase (brackets), involving the circled 5′-monophosphate ends, as revealed by RT-PCR with the indicated primers. Sequences of precursor RNA sequences are shown with

    Article Snippet: For rephosphorylation, dephosphorylated RNA was incubated at 125 ng/μl for 50 min at 37°C in RNA ligase buffer with 0.5 U of T4 polynucleotide kinase (New England Biolabs)/μl, purifying the product RNA as described above.

    Techniques: Sequencing, Ligation, Reverse Transcription Polymerase Chain Reaction

    TSS-EMOTE flowchart. The TSS-EMOTE assay consists of a wet-lab library preparation (panels a to g ) and in silico analyses (panel H to N). An asterisk continually marks the original 5’-base of tri-phosphorylated RNA (thin red line). a Total RNA is purified, and digested with XRN1 5’-exonuclease, which removes the vast majority of 5’ mono-phosphorylated RNA from the sample (including 16S and 23S rRNA). b and c The XRN1 treated RNA is mixed with large excess of a synthetic RNA oligo (Rp6, shown in blue), and split into two pools. Both pools receive T4 RNA ligase, but only the “+RppH” pool is co-treated with RppH, an enzyme that converts 5’ tri-phosphorylated ends to mono-phosphorylated ends, thus allowing the ligase to use them as substrates. d and e After the ligation reaction, a semi-random primer is used to reverse-transcribe the RNA and simultaneously add a 2.0 Illumina adapter (black “B”). This results in cDNA with a 2.0 Illumina adaptor (for reverse reads in paired-end sequencing) at the 5’-end and if the template RNA was ligated to an Rp6 oligo, then the cDNA will also have a complementary sequence to Rp6 at the 3’-end (cRp6). f PCR is used to specifically amplify cDNAs that carry the 2.0 Illumina adaptor and cRp6 sequences. This step moreover adds a 1.0 Illumina adaptor (for forward reads in paired-end sequencing) and a sample-specific 4-base EMOTE barcode (blue line and “XXX”, respectively) to index the molecules (different barcodes for the -RppH and + RppH pools). The barcode of the -RppH pool will designate molecules where the XRN1 treatments has been incomplete, and this information is incorporated into the in silico analysis (see below). g The barcoded DNA from various samples (and pools) can be mixed, and loaded directly into an Illumina HiSeq machine. Millions of 50 nt sequences are obtained, each of which will span the EMOTE barcode, both known and random sections of the Rp6 oligo (see Methods ), and it will reveal the first 20 nt of the native 5’-end of the ligated RNA molecule. These 20 nt are sufficient to map the vast majority of 5’-ends to a unique position on the small genomes of the bacteria in this study. However, longer Illumina reads (and thus longer mapping sequences) can be used if the TSSs are in repeated regions or if large-genome organisms, such as humans, are being examined. h The in silico pipeline input consists of stranded RNA-seq reads for one or multiple biological replicates in FASTQ format. Each replicate includes a FASTQ for the -RppH pool and another for the + RppH pool. i The FASTQ files go through EMOTE-conv software [ 51 ] that parses the reads, aligns them to the genome, and perform the quantification. Thus, for each genomic position we obtain the number of reads whose first nucleotide align at this genomic position, and on which strand it maps. The counts are further corrected for PCR biases by looking at the unique molecular identifiers (UMIs) sequences available in the unaligned part of the EMOTE read. j Quantification counts obtained for + RppH and -RppH pools are compared through a beta-binomial model that tests whether the identified 5’ ends in the + RppH pool is significantly enriched over the identified 5’ ends in the -RppH pool at a given position. The process results in a p-value that reflects our confidence in the genomic position to be enriched in the + RppH pool of the biological replicate. k The p-values of all the biological replicates are combined into a single p-value with Fisher’s method. l and m To correct the p-values for multiple testing across all genomic positions, the false discovery rate (FDR) is evaluated and only those with a FDR ≤ 0.01 are considered to be TSSs. Note also that for the FDR is only calculated for genomic positions with at least 5 detected ligation-events in at least one of the + RppH pools (UMI ≥ 5). n The TSSs then enter an annotation process that retrieve their surrounding sequence and downstream ORFs. TSSs separated by less than 5 bp are clustered together. Finally, to draw a global picture of operon structures, an independent detection of transcription terminators is operated with the software TransTermHP [ 39 ]. o Sequence of the RNA oligo Rp6 and a typical Illumina sequencing read from a TSS-EMOTE experiment. The Recognition Sequence serves as priming site for the PCR in panel F. UMI: The randomly incorporated nucleotides in the Rp6 oligo that serves to whether Illumina reads with identical Mapping Sequences originate from separate ligation events. CS: Control Sequence. EB: EMOTE barcode to index the Illumina reads. An asterisk indicates the 5’ nucleotide of the original RNA molecule

    Journal: BMC Genomics

    Article Title: TSS-EMOTE, a refined protocol for a more complete and less biased global mapping of transcription start sites in bacterial pathogens

    doi: 10.1186/s12864-016-3211-3

    Figure Lengend Snippet: TSS-EMOTE flowchart. The TSS-EMOTE assay consists of a wet-lab library preparation (panels a to g ) and in silico analyses (panel H to N). An asterisk continually marks the original 5’-base of tri-phosphorylated RNA (thin red line). a Total RNA is purified, and digested with XRN1 5’-exonuclease, which removes the vast majority of 5’ mono-phosphorylated RNA from the sample (including 16S and 23S rRNA). b and c The XRN1 treated RNA is mixed with large excess of a synthetic RNA oligo (Rp6, shown in blue), and split into two pools. Both pools receive T4 RNA ligase, but only the “+RppH” pool is co-treated with RppH, an enzyme that converts 5’ tri-phosphorylated ends to mono-phosphorylated ends, thus allowing the ligase to use them as substrates. d and e After the ligation reaction, a semi-random primer is used to reverse-transcribe the RNA and simultaneously add a 2.0 Illumina adapter (black “B”). This results in cDNA with a 2.0 Illumina adaptor (for reverse reads in paired-end sequencing) at the 5’-end and if the template RNA was ligated to an Rp6 oligo, then the cDNA will also have a complementary sequence to Rp6 at the 3’-end (cRp6). f PCR is used to specifically amplify cDNAs that carry the 2.0 Illumina adaptor and cRp6 sequences. This step moreover adds a 1.0 Illumina adaptor (for forward reads in paired-end sequencing) and a sample-specific 4-base EMOTE barcode (blue line and “XXX”, respectively) to index the molecules (different barcodes for the -RppH and + RppH pools). The barcode of the -RppH pool will designate molecules where the XRN1 treatments has been incomplete, and this information is incorporated into the in silico analysis (see below). g The barcoded DNA from various samples (and pools) can be mixed, and loaded directly into an Illumina HiSeq machine. Millions of 50 nt sequences are obtained, each of which will span the EMOTE barcode, both known and random sections of the Rp6 oligo (see Methods ), and it will reveal the first 20 nt of the native 5’-end of the ligated RNA molecule. These 20 nt are sufficient to map the vast majority of 5’-ends to a unique position on the small genomes of the bacteria in this study. However, longer Illumina reads (and thus longer mapping sequences) can be used if the TSSs are in repeated regions or if large-genome organisms, such as humans, are being examined. h The in silico pipeline input consists of stranded RNA-seq reads for one or multiple biological replicates in FASTQ format. Each replicate includes a FASTQ for the -RppH pool and another for the + RppH pool. i The FASTQ files go through EMOTE-conv software [ 51 ] that parses the reads, aligns them to the genome, and perform the quantification. Thus, for each genomic position we obtain the number of reads whose first nucleotide align at this genomic position, and on which strand it maps. The counts are further corrected for PCR biases by looking at the unique molecular identifiers (UMIs) sequences available in the unaligned part of the EMOTE read. j Quantification counts obtained for + RppH and -RppH pools are compared through a beta-binomial model that tests whether the identified 5’ ends in the + RppH pool is significantly enriched over the identified 5’ ends in the -RppH pool at a given position. The process results in a p-value that reflects our confidence in the genomic position to be enriched in the + RppH pool of the biological replicate. k The p-values of all the biological replicates are combined into a single p-value with Fisher’s method. l and m To correct the p-values for multiple testing across all genomic positions, the false discovery rate (FDR) is evaluated and only those with a FDR ≤ 0.01 are considered to be TSSs. Note also that for the FDR is only calculated for genomic positions with at least 5 detected ligation-events in at least one of the + RppH pools (UMI ≥ 5). n The TSSs then enter an annotation process that retrieve their surrounding sequence and downstream ORFs. TSSs separated by less than 5 bp are clustered together. Finally, to draw a global picture of operon structures, an independent detection of transcription terminators is operated with the software TransTermHP [ 39 ]. o Sequence of the RNA oligo Rp6 and a typical Illumina sequencing read from a TSS-EMOTE experiment. The Recognition Sequence serves as priming site for the PCR in panel F. UMI: The randomly incorporated nucleotides in the Rp6 oligo that serves to whether Illumina reads with identical Mapping Sequences originate from separate ligation events. CS: Control Sequence. EB: EMOTE barcode to index the Illumina reads. An asterisk indicates the 5’ nucleotide of the original RNA molecule

    Article Snippet: The “+RppH mix” consisted of 1.5 μl water, 2 ul RNA ligase buffer, 2 μl 10 mM ATP, 1 μl Murine RNase inhibitor, 2 μl RNA ligase 1, and 2 μl RppH (New England Biolabs).

    Techniques: In Silico, Purification, Ligation, Sequencing, Polymerase Chain Reaction, RNA Sequencing Assay, Software