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    Structured Review

    New England Biolabs rna ligase buffer
    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 <t>RNA</t> (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 <t>ligase,</t> but only <t>the</t> <t>“+RppH”</t> 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
    Rna Ligase Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) 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

    2) 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

    3) Product Images from "HDLBP binds ER-targeted mRNAs by multivalent interactions to promote protein synthesis of transmembrane and secreted proteins"

    Article Title: HDLBP binds ER-targeted mRNAs by multivalent interactions to promote protein synthesis of transmembrane and secreted proteins

    Journal: Nature Communications

    doi: 10.1038/s41467-022-30322-7

    HDLBP crosslinks to tRNAs decoding CU/UU-containing codons. a Difference in codon frequencies in the P-site (top) and E-site codons (bottom) in HDLBP KO vs. WT. Mean codon shift was calculated for four replicates (mean ± standard deviation is shown). b Enrichment of tRNAs in HDLBP PAR-CLIP and their binding sites. T-C transitions in tRNAs were normalized to total tRNA abundance and ranked from highest to the lowest value (left to right). For each T-C transition, we displayed its transition specificity (T-C transition vs. total read coverage). Mean values of two PAR-CLIP biological replicates ± SD are depicted. Total log2-transformed tRNA abundance and codon usage are also shown (top). c (Left) Browser representation of alignment to tRNA Leu-UAG. T-C transitions in the D-loop and V-region are indicated for the HDLBP PAR-CLIP dataset. The second track shows coverage in the total RNA sample. (Right) HDLBP crosslinked uridines are indicated with respect to secondary tRNA structure.
    Figure Legend Snippet: HDLBP crosslinks to tRNAs decoding CU/UU-containing codons. a Difference in codon frequencies in the P-site (top) and E-site codons (bottom) in HDLBP KO vs. WT. Mean codon shift was calculated for four replicates (mean ± standard deviation is shown). b Enrichment of tRNAs in HDLBP PAR-CLIP and their binding sites. T-C transitions in tRNAs were normalized to total tRNA abundance and ranked from highest to the lowest value (left to right). For each T-C transition, we displayed its transition specificity (T-C transition vs. total read coverage). Mean values of two PAR-CLIP biological replicates ± SD are depicted. Total log2-transformed tRNA abundance and codon usage are also shown (top). c (Left) Browser representation of alignment to tRNA Leu-UAG. T-C transitions in the D-loop and V-region are indicated for the HDLBP PAR-CLIP dataset. The second track shows coverage in the total RNA sample. (Right) HDLBP crosslinked uridines are indicated with respect to secondary tRNA structure.

    Techniques Used: Standard Deviation, Cross-linking Immunoprecipitation, Binding Assay, Transformation Assay

    HDLBP interacts with the translational apparatus. a PAR-CLIP coverage and crosslinks detected in pre-rRNA regions. For comparison, results for IRE1 PAR-CLIP are included. Expansion segment positions are indicated in green. b Structures of the human 80S ribosome (PDB: 4V6X) and the SRP-ribosome complex (PDB: 3JAJ) were juxtaposed and HDLBP rRNA and 7SL RNA crosslinked nucleotides were mapped (indicated in red). c RNA immunoprecipitation was performed with FLAG/HA-HDLBP as bait. Co-precipitating RNAs were detected by qRT-PCR. Average fold enrichment (anti-FLAG vs. IgG control) from four replicates was calculated with error bars indicating standard deviation. Results are shown for 7SL RNA, IGF2R, YWHAZ, CD46, and ATP1A1 are shown, along with the mtDNA-encoded mRNA (MT-CO1) as a negative control. d BioID analysis of proteins in proximity to BirA-FLAG-HDLBP. The top 60 enriched proteins (LFQ(Dox) vs. LFQ(noDox) > 3) were ranked according to mean LFQ (three replicates of Dox samples). The size of the dot corresponds to the enrichment value. e Gene Ontology enrichment analysis of 249 enriched BioID proteins. Adjusted p values for the top five enriched categories are shown. f FLAG/HA-HDLBP was co-immunoprecipitated with either anti-FLAG or IgG antibodies. Western analysis of input lysates (0.25%) and eluates (19%) was performed with antibodies as indicated. a – f Source data are provided as a Source Data file.
    Figure Legend Snippet: HDLBP interacts with the translational apparatus. a PAR-CLIP coverage and crosslinks detected in pre-rRNA regions. For comparison, results for IRE1 PAR-CLIP are included. Expansion segment positions are indicated in green. b Structures of the human 80S ribosome (PDB: 4V6X) and the SRP-ribosome complex (PDB: 3JAJ) were juxtaposed and HDLBP rRNA and 7SL RNA crosslinked nucleotides were mapped (indicated in red). c RNA immunoprecipitation was performed with FLAG/HA-HDLBP as bait. Co-precipitating RNAs were detected by qRT-PCR. Average fold enrichment (anti-FLAG vs. IgG control) from four replicates was calculated with error bars indicating standard deviation. Results are shown for 7SL RNA, IGF2R, YWHAZ, CD46, and ATP1A1 are shown, along with the mtDNA-encoded mRNA (MT-CO1) as a negative control. d BioID analysis of proteins in proximity to BirA-FLAG-HDLBP. The top 60 enriched proteins (LFQ(Dox) vs. LFQ(noDox) > 3) were ranked according to mean LFQ (three replicates of Dox samples). The size of the dot corresponds to the enrichment value. e Gene Ontology enrichment analysis of 249 enriched BioID proteins. Adjusted p values for the top five enriched categories are shown. f FLAG/HA-HDLBP was co-immunoprecipitated with either anti-FLAG or IgG antibodies. Western analysis of input lysates (0.25%) and eluates (19%) was performed with antibodies as indicated. a – f Source data are provided as a Source Data file.

    Techniques Used: Cross-linking Immunoprecipitation, Immunoprecipitation, Quantitative RT-PCR, Standard Deviation, Negative Control, Western Blot

    HDLBP specificity for membrane-bound mRNAs. a Frequency of top ten HDLBP crosslinked seven-mers located either in 3′ UTR or CDS of membrane-bound and cytosolic mRNAs. b Sequence logo of top five HDLBP crosslinked seven-mers ranked according to their frequency among all detected crosslinked seven-mers. c Distribution of z -scores calculated from differences in the frequency of all possible k-mers within membrane-bound and cytosolic CDS and 3′ UTR sequences. For each k-mer length, this analysis was performed for the group of top 40 HDLBP crosslinked k-mers (left) and all other k-mers (right). d p values of pairwise Wilcoxon rank-sum test between z -scores obtained for top 40 bound HDLBP k-mers as described in ( c ). e HDLBP multivalency analysis in +40/−40 nt regions around cross-linking sites. To evaluate the T-C binding affinity as a function of multivalency of HDLBP binding sites, we binned the multivalency scores for the top ten enriched four-mers within the 40-nt regions into five categories (group sized from highest to lowest score groups, n = 994, n = 973, n = 673, n = 1036, n = 1308). The total normalized T-C transition signal over the +40/−40 nt regions was then plotted for all five categories. Lower and upper hinges of box plots correspond to the 25th and 75th percentiles, respectively. Upper and lower whiskers extend from the hinge to the largest or smallest value no further than the 1.5× interquartile range from the hinge, respectively. Center lines of box plots depict the median values. f Analysis of the percentage of total T-C transitions for every nucleotide position within the +40/−40 nt region for each multivalency bin. The two bins with the highest multivalency scores are shown and correspond to ( e ). g Comparison of mean multivalency scores between differentially localized mRNAs in their CDS. A positive set (a four-mer group consisting of top ten HDLBP crosslinked four-mers, UUCU) and a negative set (AAGU) with no HDLBP enrichment. Occurrence of these four-mer groups were counted in 30-nt sliding windows and the mean score per transcript was computed. Mean distribution was then compared between different localized CDS by Wilcoxon rank-sum tests. h Apparent dissociation constants of recombinant GST-HDLBP fragments (constructs A though D) and full-length protein (FL), schematically shown, for different RNA oligonucleotides as determined by fluorescence anisotropy binding assays. Dissociation constants that were measured but could not be determined are indicated with “n.d.” and interaction that were not measured by “—“. i Fluorescence anisotropy binding assays for RNA oligonucleotides with different number of HDLBP binding four-mers (left, H40-44). GST-HDLBP construct B (KH5-9) was incubated with FAM-labeled RNA oligonucleotides, anisotropy measured and K D determined from the binding curves (middle panel). For each oligonucleotide, three independent K d values were determined. Significant differences in K d values were evaluated using two-sided t -test and are indicated with asterisks (* P
    Figure Legend Snippet: HDLBP specificity for membrane-bound mRNAs. a Frequency of top ten HDLBP crosslinked seven-mers located either in 3′ UTR or CDS of membrane-bound and cytosolic mRNAs. b Sequence logo of top five HDLBP crosslinked seven-mers ranked according to their frequency among all detected crosslinked seven-mers. c Distribution of z -scores calculated from differences in the frequency of all possible k-mers within membrane-bound and cytosolic CDS and 3′ UTR sequences. For each k-mer length, this analysis was performed for the group of top 40 HDLBP crosslinked k-mers (left) and all other k-mers (right). d p values of pairwise Wilcoxon rank-sum test between z -scores obtained for top 40 bound HDLBP k-mers as described in ( c ). e HDLBP multivalency analysis in +40/−40 nt regions around cross-linking sites. To evaluate the T-C binding affinity as a function of multivalency of HDLBP binding sites, we binned the multivalency scores for the top ten enriched four-mers within the 40-nt regions into five categories (group sized from highest to lowest score groups, n = 994, n = 973, n = 673, n = 1036, n = 1308). The total normalized T-C transition signal over the +40/−40 nt regions was then plotted for all five categories. Lower and upper hinges of box plots correspond to the 25th and 75th percentiles, respectively. Upper and lower whiskers extend from the hinge to the largest or smallest value no further than the 1.5× interquartile range from the hinge, respectively. Center lines of box plots depict the median values. f Analysis of the percentage of total T-C transitions for every nucleotide position within the +40/−40 nt region for each multivalency bin. The two bins with the highest multivalency scores are shown and correspond to ( e ). g Comparison of mean multivalency scores between differentially localized mRNAs in their CDS. A positive set (a four-mer group consisting of top ten HDLBP crosslinked four-mers, UUCU) and a negative set (AAGU) with no HDLBP enrichment. Occurrence of these four-mer groups were counted in 30-nt sliding windows and the mean score per transcript was computed. Mean distribution was then compared between different localized CDS by Wilcoxon rank-sum tests. h Apparent dissociation constants of recombinant GST-HDLBP fragments (constructs A though D) and full-length protein (FL), schematically shown, for different RNA oligonucleotides as determined by fluorescence anisotropy binding assays. Dissociation constants that were measured but could not be determined are indicated with “n.d.” and interaction that were not measured by “—“. i Fluorescence anisotropy binding assays for RNA oligonucleotides with different number of HDLBP binding four-mers (left, H40-44). GST-HDLBP construct B (KH5-9) was incubated with FAM-labeled RNA oligonucleotides, anisotropy measured and K D determined from the binding curves (middle panel). For each oligonucleotide, three independent K d values were determined. Significant differences in K d values were evaluated using two-sided t -test and are indicated with asterisks (* P

    Techniques Used: Sequencing, Binding Assay, Recombinant, Construct, Fluorescence, Incubation, Labeling

    4) Product Images from "HDLBP binds ER-targeted mRNAs by multivalent interactions to promote protein synthesis of transmembrane and secreted proteins"

    Article Title: HDLBP binds ER-targeted mRNAs by multivalent interactions to promote protein synthesis of transmembrane and secreted proteins

    Journal: Nature Communications

    doi: 10.1038/s41467-022-30322-7

    HDLBP crosslinks to tRNAs decoding CU/UU-containing codons. a Difference in codon frequencies in the P-site (top) and E-site codons (bottom) in HDLBP KO vs. WT. Mean codon shift was calculated for four replicates (mean ± standard deviation is shown). b Enrichment of tRNAs in HDLBP PAR-CLIP and their binding sites. T-C transitions in tRNAs were normalized to total tRNA abundance and ranked from highest to the lowest value (left to right). For each T-C transition, we displayed its transition specificity (T-C transition vs. total read coverage). Mean values of two PAR-CLIP biological replicates ± SD are depicted. Total log2-transformed tRNA abundance and codon usage are also shown (top). c (Left) Browser representation of alignment to tRNA Leu-UAG. T-C transitions in the D-loop and V-region are indicated for the HDLBP PAR-CLIP dataset. The second track shows coverage in the total RNA sample. (Right) HDLBP crosslinked uridines are indicated with respect to secondary tRNA structure.
    Figure Legend Snippet: HDLBP crosslinks to tRNAs decoding CU/UU-containing codons. a Difference in codon frequencies in the P-site (top) and E-site codons (bottom) in HDLBP KO vs. WT. Mean codon shift was calculated for four replicates (mean ± standard deviation is shown). b Enrichment of tRNAs in HDLBP PAR-CLIP and their binding sites. T-C transitions in tRNAs were normalized to total tRNA abundance and ranked from highest to the lowest value (left to right). For each T-C transition, we displayed its transition specificity (T-C transition vs. total read coverage). Mean values of two PAR-CLIP biological replicates ± SD are depicted. Total log2-transformed tRNA abundance and codon usage are also shown (top). c (Left) Browser representation of alignment to tRNA Leu-UAG. T-C transitions in the D-loop and V-region are indicated for the HDLBP PAR-CLIP dataset. The second track shows coverage in the total RNA sample. (Right) HDLBP crosslinked uridines are indicated with respect to secondary tRNA structure.

    Techniques Used: Standard Deviation, Cross-linking Immunoprecipitation, Binding Assay, Transformation Assay

    HDLBP interacts with the translational apparatus. a PAR-CLIP coverage and crosslinks detected in pre-rRNA regions. For comparison, results for IRE1 PAR-CLIP are included. Expansion segment positions are indicated in green. b Structures of the human 80S ribosome (PDB: 4V6X) and the SRP-ribosome complex (PDB: 3JAJ) were juxtaposed and HDLBP rRNA and 7SL RNA crosslinked nucleotides were mapped (indicated in red). c RNA immunoprecipitation was performed with FLAG/HA-HDLBP as bait. Co-precipitating RNAs were detected by qRT-PCR. Average fold enrichment (anti-FLAG vs. IgG control) from four replicates was calculated with error bars indicating standard deviation. Results are shown for 7SL RNA, IGF2R, YWHAZ, CD46, and ATP1A1 are shown, along with the mtDNA-encoded mRNA (MT-CO1) as a negative control. d BioID analysis of proteins in proximity to BirA-FLAG-HDLBP. The top 60 enriched proteins (LFQ(Dox) vs. LFQ(noDox) > 3) were ranked according to mean LFQ (three replicates of Dox samples). The size of the dot corresponds to the enrichment value. e Gene Ontology enrichment analysis of 249 enriched BioID proteins. Adjusted p values for the top five enriched categories are shown. f FLAG/HA-HDLBP was co-immunoprecipitated with either anti-FLAG or IgG antibodies. Western analysis of input lysates (0.25%) and eluates (19%) was performed with antibodies as indicated. a – f Source data are provided as a Source Data file.
    Figure Legend Snippet: HDLBP interacts with the translational apparatus. a PAR-CLIP coverage and crosslinks detected in pre-rRNA regions. For comparison, results for IRE1 PAR-CLIP are included. Expansion segment positions are indicated in green. b Structures of the human 80S ribosome (PDB: 4V6X) and the SRP-ribosome complex (PDB: 3JAJ) were juxtaposed and HDLBP rRNA and 7SL RNA crosslinked nucleotides were mapped (indicated in red). c RNA immunoprecipitation was performed with FLAG/HA-HDLBP as bait. Co-precipitating RNAs were detected by qRT-PCR. Average fold enrichment (anti-FLAG vs. IgG control) from four replicates was calculated with error bars indicating standard deviation. Results are shown for 7SL RNA, IGF2R, YWHAZ, CD46, and ATP1A1 are shown, along with the mtDNA-encoded mRNA (MT-CO1) as a negative control. d BioID analysis of proteins in proximity to BirA-FLAG-HDLBP. The top 60 enriched proteins (LFQ(Dox) vs. LFQ(noDox) > 3) were ranked according to mean LFQ (three replicates of Dox samples). The size of the dot corresponds to the enrichment value. e Gene Ontology enrichment analysis of 249 enriched BioID proteins. Adjusted p values for the top five enriched categories are shown. f FLAG/HA-HDLBP was co-immunoprecipitated with either anti-FLAG or IgG antibodies. Western analysis of input lysates (0.25%) and eluates (19%) was performed with antibodies as indicated. a – f Source data are provided as a Source Data file.

    Techniques Used: Cross-linking Immunoprecipitation, Immunoprecipitation, Quantitative RT-PCR, Standard Deviation, Negative Control, Western Blot

    HDLBP specificity for membrane-bound mRNAs. a Frequency of top ten HDLBP crosslinked seven-mers located either in 3′ UTR or CDS of membrane-bound and cytosolic mRNAs. b Sequence logo of top five HDLBP crosslinked seven-mers ranked according to their frequency among all detected crosslinked seven-mers. c Distribution of z -scores calculated from differences in the frequency of all possible k-mers within membrane-bound and cytosolic CDS and 3′ UTR sequences. For each k-mer length, this analysis was performed for the group of top 40 HDLBP crosslinked k-mers (left) and all other k-mers (right). d p values of pairwise Wilcoxon rank-sum test between z -scores obtained for top 40 bound HDLBP k-mers as described in ( c ). e HDLBP multivalency analysis in +40/−40 nt regions around cross-linking sites. To evaluate the T-C binding affinity as a function of multivalency of HDLBP binding sites, we binned the multivalency scores for the top ten enriched four-mers within the 40-nt regions into five categories (group sized from highest to lowest score groups, n = 994, n = 973, n = 673, n = 1036, n = 1308). The total normalized T-C transition signal over the +40/−40 nt regions was then plotted for all five categories. Lower and upper hinges of box plots correspond to the 25th and 75th percentiles, respectively. Upper and lower whiskers extend from the hinge to the largest or smallest value no further than the 1.5× interquartile range from the hinge, respectively. Center lines of box plots depict the median values. f Analysis of the percentage of total T-C transitions for every nucleotide position within the +40/−40 nt region for each multivalency bin. The two bins with the highest multivalency scores are shown and correspond to ( e ). g Comparison of mean multivalency scores between differentially localized mRNAs in their CDS. A positive set (a four-mer group consisting of top ten HDLBP crosslinked four-mers, UUCU) and a negative set (AAGU) with no HDLBP enrichment. Occurrence of these four-mer groups were counted in 30-nt sliding windows and the mean score per transcript was computed. Mean distribution was then compared between different localized CDS by Wilcoxon rank-sum tests. h Apparent dissociation constants of recombinant GST-HDLBP fragments (constructs A though D) and full-length protein (FL), schematically shown, for different RNA oligonucleotides as determined by fluorescence anisotropy binding assays. Dissociation constants that were measured but could not be determined are indicated with “n.d.” and interaction that were not measured by “—“. i Fluorescence anisotropy binding assays for RNA oligonucleotides with different number of HDLBP binding four-mers (left, H40-44). GST-HDLBP construct B (KH5-9) was incubated with FAM-labeled RNA oligonucleotides, anisotropy measured and K D determined from the binding curves (middle panel). For each oligonucleotide, three independent K d values were determined. Significant differences in K d values were evaluated using two-sided t -test and are indicated with asterisks (* P
    Figure Legend Snippet: HDLBP specificity for membrane-bound mRNAs. a Frequency of top ten HDLBP crosslinked seven-mers located either in 3′ UTR or CDS of membrane-bound and cytosolic mRNAs. b Sequence logo of top five HDLBP crosslinked seven-mers ranked according to their frequency among all detected crosslinked seven-mers. c Distribution of z -scores calculated from differences in the frequency of all possible k-mers within membrane-bound and cytosolic CDS and 3′ UTR sequences. For each k-mer length, this analysis was performed for the group of top 40 HDLBP crosslinked k-mers (left) and all other k-mers (right). d p values of pairwise Wilcoxon rank-sum test between z -scores obtained for top 40 bound HDLBP k-mers as described in ( c ). e HDLBP multivalency analysis in +40/−40 nt regions around cross-linking sites. To evaluate the T-C binding affinity as a function of multivalency of HDLBP binding sites, we binned the multivalency scores for the top ten enriched four-mers within the 40-nt regions into five categories (group sized from highest to lowest score groups, n = 994, n = 973, n = 673, n = 1036, n = 1308). The total normalized T-C transition signal over the +40/−40 nt regions was then plotted for all five categories. Lower and upper hinges of box plots correspond to the 25th and 75th percentiles, respectively. Upper and lower whiskers extend from the hinge to the largest or smallest value no further than the 1.5× interquartile range from the hinge, respectively. Center lines of box plots depict the median values. f Analysis of the percentage of total T-C transitions for every nucleotide position within the +40/−40 nt region for each multivalency bin. The two bins with the highest multivalency scores are shown and correspond to ( e ). g Comparison of mean multivalency scores between differentially localized mRNAs in their CDS. A positive set (a four-mer group consisting of top ten HDLBP crosslinked four-mers, UUCU) and a negative set (AAGU) with no HDLBP enrichment. Occurrence of these four-mer groups were counted in 30-nt sliding windows and the mean score per transcript was computed. Mean distribution was then compared between different localized CDS by Wilcoxon rank-sum tests. h Apparent dissociation constants of recombinant GST-HDLBP fragments (constructs A though D) and full-length protein (FL), schematically shown, for different RNA oligonucleotides as determined by fluorescence anisotropy binding assays. Dissociation constants that were measured but could not be determined are indicated with “n.d.” and interaction that were not measured by “—“. i Fluorescence anisotropy binding assays for RNA oligonucleotides with different number of HDLBP binding four-mers (left, H40-44). GST-HDLBP construct B (KH5-9) was incubated with FAM-labeled RNA oligonucleotides, anisotropy measured and K D determined from the binding curves (middle panel). For each oligonucleotide, three independent K d values were determined. Significant differences in K d values were evaluated using two-sided t -test and are indicated with asterisks (* P

    Techniques Used: Sequencing, Binding Assay, Recombinant, Construct, Fluorescence, Incubation, Labeling

    5) 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

    6) 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

    7) 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

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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: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Detection of sPom121 mRNA and protein expression in human cells. ( A ) Schematic of two putative <t>Pom121</t> 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 <t>RNA</t> 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).
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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

    Post-transcriptional processing of lncCOBRA1. (A) Diagram of lncCOBRA1 ( AT1G05913 ) locus. Gray arrows represent the two snoRNAs annotated within lncCOBRA1. Red arrows represent the two primers used for 5′ RACE and red triangle represents the 5′ end identified by 5′ RACE PCR in (B) . Blue arrow represents the primer used for 3′ RACE. Blue triangles represent the 3’ most end identified through Sanger sequencing 14 colonies. (B) Three biological replicates of 5′ RACE with primers indicated in (B) . Red triangles represent the two major bands of PCR product. Ladder is 1 kb + . (C) PCR results from 3′ RACE in Col-0 5-day-old seedlings. –/+T4 RNA ligase, –/+SuperScript II. Ladder is 1 kb +.

    Journal: Frontiers in Plant Science

    Article Title: A Conserved Long Intergenic Non-coding RNA Containing snoRNA Sequences, lncCOBRA1, Affects Arabidopsis Germination and Development

    doi: 10.3389/fpls.2022.906603

    Figure Lengend Snippet: Post-transcriptional processing of lncCOBRA1. (A) Diagram of lncCOBRA1 ( AT1G05913 ) locus. Gray arrows represent the two snoRNAs annotated within lncCOBRA1. Red arrows represent the two primers used for 5′ RACE and red triangle represents the 5′ end identified by 5′ RACE PCR in (B) . Blue arrow represents the primer used for 3′ RACE. Blue triangles represent the 3’ most end identified through Sanger sequencing 14 colonies. (B) Three biological replicates of 5′ RACE with primers indicated in (B) . Red triangles represent the two major bands of PCR product. Ladder is 1 kb + . (C) PCR results from 3′ RACE in Col-0 5-day-old seedlings. –/+T4 RNA ligase, –/+SuperScript II. Ladder is 1 kb +.

    Article Snippet: To the 10 μL RppH reaction, we added 1 μL of 5′ RNA adapter (25 μM; RA5; 5′-GUUCAGAGUUCUACAGUCCGACGAUC-3′) that was first heated to 70°C for 2 min followed by 2 min on ice to relieve secondary structures, 1 μL 10 mM ATP (New England BioLabs; Ipswitch, MA, United States), 10 units T4 RNA Ligase 1 (New England BioLabs; Ipswitch, MA, United States), 1 μL T4 RNA Ligase Buffer (New England BioLabs; Ipswitch, MA, United States), and 40 units RNaseOUT (Invitrogen; Carlsbad, CA, United States) and incubated for 3 h at 20°C followed by an overnight ethanol precipitated.

    Techniques: Polymerase Chain Reaction, Sequencing

    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.

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0101812

    Figure Lengend 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.

    Article Snippet: The products were then treated with T4 Polynucleotide Kinase to add mono-phosphate to non-capped mRNA to ready it for ligation; a reaction mixture consisting of 1 µl of T4 Polynucleotide Kinase (Fermentas, # EK0032), 2 µl of RNA Ligase Reaction Buffer (New England Biolabs), 0.5 µl of RNaseOUT (Invitrogen, #10777-019), 1 µl of 100 mM ATP solution (Fermentas, #R0441), and 15.5 µl of alkaline phosphatase-treated RNA was incubated for 30 minutes at 37°C.

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

    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).

    Journal: Genes & Development

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

    doi: 10.1101/gad.280941.116

    Figure Lengend 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).

    Article Snippet: Next, an anchor primer was ligated to the 3′ end of the Pom121 cDNA products (4 µL of Pom121 cDNA, 2 µL of phosphorylated anchor primer, 2 µL of RNA ligase buffer, 8 µL of 50% PEG 8000, 1 µL of 10 mM ATP, 1 µL of 0.1 M DTT, 1 µL of SS RNA ligase 1 [New England Biolabs]) overnight at 25°C.

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