pom121 cdna  (New England Biolabs)


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    New England Biolabs pom121 cdna
    Monarch PCR and DNA Cleanup Kit
    Monarch PCR and DNA Cleanup Kit 250 preps
    https://www.bioz.com/result/pom121 cdna/product/New England Biolabs
    Average 90 stars, based on 1341 article reviews
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    pom121 cdna - by Bioz Stars, 2020-08
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    Images

    1) 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

    2) Product Images from "Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis"

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky067

    Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).
    Figure Legend Snippet: Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).

    Techniques Used: Plasmid Preparation, Isolation, Purification, Polymerase Chain Reaction, Clone Assay

    Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).
    Figure Legend Snippet: Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).

    Techniques Used: Plasmid Preparation, Amplification, Polymerase Chain Reaction, Clone Assay

    Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.
    Figure Legend Snippet: Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.

    Techniques Used: Sequencing, Polymerase Chain Reaction

    3) Product Images from "Uridylation by TUT4/7 Restricts Retrotransposition of Human LINE-1s"

    Article Title: Uridylation by TUT4/7 Restricts Retrotransposition of Human LINE-1s

    Journal: Cell

    doi: 10.1016/j.cell.2018.07.022

    Graphical Visualization of the 3′ RACE-Seq Approach, Related to Figure 2 (A) Graphical representation of 3′ RACE-seq library preparation and the oligonucleotides used. First, the 3′ adaptor RA3_15N was joined to the 3′ end of RNA by enzymatic ligation. The adaptor has: (i) 5′ rApp modification for efficient and specific ligation by the truncated T4 RNA ligase 2, (ii) delimiter sequence to be used in bioinformatics analyses to exclude RT and PCR artifacts (CTGAC, highlighted in violet), (iii) unique 15N barcode for individual transcript barcoding (highlighted in green), (iv) anchor sequence to pair with the reverse transcription primer (underlined) and (v) dideoxyC on the 3′ end to prevent concatamer formation. The RNA ligated to the adaptor sequence was purified from excess adaptor and reverse transcription was performed with the RT primer, which is compatible with Illumina sequencing and has: (i) sequences to base-pair with the adaptor (underlined), (ii) 6-nucleotide barcode for sample barcoding (highlighted in red), (iii) sequences that base pair with the universal outer primer for nested PCR (blue). Libraries were generated by nested PCR with 2 outer forward primers (F1 and F2) and a single universal reverse primer (uni rev). PCR amplicons of first and second PCRs were purified from excess primers on AmPure beads (Agencourt) before beginning the next step. (B) Flowchart of the bioinformatics approach to 3′ RACE-seq data analysis. The procedure starts at the top. Datasets are shown in rectangles. Software used is depicted in hexagons.
    Figure Legend Snippet: Graphical Visualization of the 3′ RACE-Seq Approach, Related to Figure 2 (A) Graphical representation of 3′ RACE-seq library preparation and the oligonucleotides used. First, the 3′ adaptor RA3_15N was joined to the 3′ end of RNA by enzymatic ligation. The adaptor has: (i) 5′ rApp modification for efficient and specific ligation by the truncated T4 RNA ligase 2, (ii) delimiter sequence to be used in bioinformatics analyses to exclude RT and PCR artifacts (CTGAC, highlighted in violet), (iii) unique 15N barcode for individual transcript barcoding (highlighted in green), (iv) anchor sequence to pair with the reverse transcription primer (underlined) and (v) dideoxyC on the 3′ end to prevent concatamer formation. The RNA ligated to the adaptor sequence was purified from excess adaptor and reverse transcription was performed with the RT primer, which is compatible with Illumina sequencing and has: (i) sequences to base-pair with the adaptor (underlined), (ii) 6-nucleotide barcode for sample barcoding (highlighted in red), (iii) sequences that base pair with the universal outer primer for nested PCR (blue). Libraries were generated by nested PCR with 2 outer forward primers (F1 and F2) and a single universal reverse primer (uni rev). PCR amplicons of first and second PCRs were purified from excess primers on AmPure beads (Agencourt) before beginning the next step. (B) Flowchart of the bioinformatics approach to 3′ RACE-seq data analysis. The procedure starts at the top. Datasets are shown in rectangles. Software used is depicted in hexagons.

    Techniques Used: Ligation, Modification, Sequencing, Polymerase Chain Reaction, Purification, Nested PCR, Generated, Software

    4) Product Images from "Cellular reagents for diagnostics and synthetic biology"

    Article Title: Cellular reagents for diagnostics and synthetic biology

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0201681

    PCR and Gibson assembly using cellular reagents. (a) Schematic depicting cellular PCR followed by cellular Gibson assembly for constructing new plasmids. Bacteria harboring target plasmids are mixed with polymerase-expressing cellular reagents and PCR is initiated by adding appropriate primers, buffer, and dNTP. The resulting PCR products are incubated with cellular reagents expressing Gibson assembly enzymes–Taq DNA polymerase, Taq DNA ligase, and T5 exonuclease–to assemble the new construct. (b) Cellular PCR amplification of vector and insert fragments directly from  E .  coli  bacteria bearing target DNA plasmids using 2 x 10 7  cells of Phusion cellular reagents. Assembly parts include: (i) “pATetO 6XHis full length” vector for two part assembly with Kan r  cassette bearing appropriate overlapping ends, and (ii) “pUC19 Fragments 1 and 2” for three part assembly with Kan r  cassette whose ends overlap with pUC19 vector fragments. (c) Gibson assembly of agarose gel purified and unpurified cellular PCR products using pure or cellular Gibson assembly reagents. In “negative control” samples the PCR products were incubated in Gibson reaction buffer without pure or cellular Gibson enzymes. “pATetO 6XHis + Kan r ”represents a two part Gibson assembly while “Puc19 Fragment 1 + pUC19 Fragment 2 + Kan r ” represents a three-part Gibson assembly. Representative number of clones recovered in each case in the presence of both ampicillin and kanamycin are reported.
    Figure Legend Snippet: PCR and Gibson assembly using cellular reagents. (a) Schematic depicting cellular PCR followed by cellular Gibson assembly for constructing new plasmids. Bacteria harboring target plasmids are mixed with polymerase-expressing cellular reagents and PCR is initiated by adding appropriate primers, buffer, and dNTP. The resulting PCR products are incubated with cellular reagents expressing Gibson assembly enzymes–Taq DNA polymerase, Taq DNA ligase, and T5 exonuclease–to assemble the new construct. (b) Cellular PCR amplification of vector and insert fragments directly from E . coli bacteria bearing target DNA plasmids using 2 x 10 7 cells of Phusion cellular reagents. Assembly parts include: (i) “pATetO 6XHis full length” vector for two part assembly with Kan r cassette bearing appropriate overlapping ends, and (ii) “pUC19 Fragments 1 and 2” for three part assembly with Kan r cassette whose ends overlap with pUC19 vector fragments. (c) Gibson assembly of agarose gel purified and unpurified cellular PCR products using pure or cellular Gibson assembly reagents. In “negative control” samples the PCR products were incubated in Gibson reaction buffer without pure or cellular Gibson enzymes. “pATetO 6XHis + Kan r ”represents a two part Gibson assembly while “Puc19 Fragment 1 + pUC19 Fragment 2 + Kan r ” represents a three-part Gibson assembly. Representative number of clones recovered in each case in the presence of both ampicillin and kanamycin are reported.

    Techniques Used: Polymerase Chain Reaction, Expressing, Incubation, Construct, Amplification, Plasmid Preparation, Agarose Gel Electrophoresis, Purification, Clone Assay

    5) Product Images from "Chimeric Phage Nanoparticles for Rapid Characterization of Bacterial Pathogens: Detection in Complex Biological Samples and Determination of Antibiotic Sensitivity"

    Article Title: Chimeric Phage Nanoparticles for Rapid Characterization of Bacterial Pathogens: Detection in Complex Biological Samples and Determination of Antibiotic Sensitivity

    Journal: ACS Sensors

    doi: 10.1021/acssensors.0c00654

    Determination of growth in the presence of antibiotics using thiolated M13KE phage and AuNPs. (a–c) Digital photos and (d–f) UV–vis spectra are shown. Samples in (a, d), (b, e), and (c, f) were grown with ampicillin, kanamycin, or tetracycline, respectively. Samples from left to right in each photo are AuNPs with no bacteria or phages, control (10 6 CFU cells with unmodified M13KE phage and AuNPs), and thiolated M13KE phage and AuNPs with the bacterial sample at the following dilutions: 1-, 10-, 10 2 -, 10 3 -, 10 4 -, 10 5 -, 10 6 -, and 10 7 -fold.
    Figure Legend Snippet: Determination of growth in the presence of antibiotics using thiolated M13KE phage and AuNPs. (a–c) Digital photos and (d–f) UV–vis spectra are shown. Samples in (a, d), (b, e), and (c, f) were grown with ampicillin, kanamycin, or tetracycline, respectively. Samples from left to right in each photo are AuNPs with no bacteria or phages, control (10 6 CFU cells with unmodified M13KE phage and AuNPs), and thiolated M13KE phage and AuNPs with the bacterial sample at the following dilutions: 1-, 10-, 10 2 -, 10 3 -, 10 4 -, 10 5 -, 10 6 -, and 10 7 -fold.

    Techniques Used:

    6) Product Images from "Ribozyme-catalysed RNA synthesis using triplet building blocks"

    Article Title: Ribozyme-catalysed RNA synthesis using triplet building blocks

    Journal: eLife

    doi: 10.7554/eLife.35255

    Ribozyme segment synthesis with random substrate pools. For maximum self-synthesis yield, we had used specific triplet substrate sets. Here we compare synthesis by 0.5 µM t5 +1 of the five t5 ‘+’ segments (30 days in −7˚C ice) using specific triplets vs. random triplet pools or reduced G-content random triplet pools. Reactions included 5 µM of each triplet in specific triplet sets (‘tri’, as in Figure 6a ‘ + ’ syntheses), random ppp NNN, or a low-G ppp NNN. The low-G ppp NNN had the same overall triplet concentration as ppp NNN, but individual triplets were five-fold less common for each constituent G (~10/2/0.4/0.08 µM for 0/1/2/3 Gs per triplet; some primer/template (P/T) concentrations were reduced here to ensure excess substrate over template). Synthesis is compared with and without some longer oligonucleotide substrates (indicated below, equimolar to template sites, replacing the corresponding triplets in ‘tri’ substrate mixes). Importantly, for all ‘+’ strand segments, full-length products are generated using ppp NNN with yields approaching those obtained when using specific triplets (calculated by densitometry, above the gel); intriguingly the low-G ppp NNN pools often gave superior yields, possibly helped by the higher concentrations of weaker-binding AU-rich triplets therein.
    Figure Legend Snippet: Ribozyme segment synthesis with random substrate pools. For maximum self-synthesis yield, we had used specific triplet substrate sets. Here we compare synthesis by 0.5 µM t5 +1 of the five t5 ‘+’ segments (30 days in −7˚C ice) using specific triplets vs. random triplet pools or reduced G-content random triplet pools. Reactions included 5 µM of each triplet in specific triplet sets (‘tri’, as in Figure 6a ‘ + ’ syntheses), random ppp NNN, or a low-G ppp NNN. The low-G ppp NNN had the same overall triplet concentration as ppp NNN, but individual triplets were five-fold less common for each constituent G (~10/2/0.4/0.08 µM for 0/1/2/3 Gs per triplet; some primer/template (P/T) concentrations were reduced here to ensure excess substrate over template). Synthesis is compared with and without some longer oligonucleotide substrates (indicated below, equimolar to template sites, replacing the corresponding triplets in ‘tri’ substrate mixes). Importantly, for all ‘+’ strand segments, full-length products are generated using ppp NNN with yields approaching those obtained when using specific triplets (calculated by densitometry, above the gel); intriguingly the low-G ppp NNN pools often gave superior yields, possibly helped by the higher concentrations of weaker-binding AU-rich triplets therein.

    Techniques Used: Concentration Assay, Generated, Binding Assay

    Type 1 enhancement of parental ribozymes. Left, transplanting the conserved 5’ ‘cap+’ sequence from types 2–6 allows the Zcore but not the 0core ribozyme triplet polymerase activity to be enhanced by type 1; shown are PAGE of extensions of primer A10 on template CCCMisAUG by the indicated ribozyme cores (5 μM of ppp AUG and ppp CCC, 0.5 μM of each RNA with type 1 annealed and added separately, −7˚C in ice for 22 hr). Right, RNA polymerase activity of Z RPR using NTPs is enhanced by type 1 addition, but only when modified with the 5’ ‘cap+’ sequence (Z RPR cap+ ), extending primer A10 on tethered template HTI (0.5 μM of each RNA, 4 mM of each NTP, at 4˚C for 68 hr).
    Figure Legend Snippet: Type 1 enhancement of parental ribozymes. Left, transplanting the conserved 5’ ‘cap+’ sequence from types 2–6 allows the Zcore but not the 0core ribozyme triplet polymerase activity to be enhanced by type 1; shown are PAGE of extensions of primer A10 on template CCCMisAUG by the indicated ribozyme cores (5 μM of ppp AUG and ppp CCC, 0.5 μM of each RNA with type 1 annealed and added separately, −7˚C in ice for 22 hr). Right, RNA polymerase activity of Z RPR using NTPs is enhanced by type 1 addition, but only when modified with the 5’ ‘cap+’ sequence (Z RPR cap+ ), extending primer A10 on tethered template HTI (0.5 μM of each RNA, 4 mM of each NTP, at 4˚C for 68 hr).

    Techniques Used: Sequencing, Activity Assay, Polyacrylamide Gel Electrophoresis, Countercurrent Chromatography, Modification

    Clonal versus polyclonal activity. Type 1 and 2 RNAs, and the indicated polyclonal ribozyme pools late in the selection, were transcribed as selection constructs generated with Tri8AUAM (then annealed to primer A10). Their abilities to incorporate eight ppp AUA triplets then self-ligate in this selection construct context were compared (left, 0.1 μM each RNA, 2 μM ppp AUA, in −7˚C ice for 5 days; right, 0.2 μM each RNA, 2 μM ppp AUA and ppp AUG 3’d , in −7˚C ice for 9 days). Left: Despite being the most active type ( Figure 2b ), type two activity fell far short of the round 19 pool polyclonal activity. Right: The incorporation of wobble pairing 3’-deoxy ‘terminator’ triplet ppp AUG 3’d (see results section on fidelity, yielding a faster-migrating band than incorporation of the cognate ppp AUA triplet) was decreased sharply after round 19, as calculated by densitometry and averaging amongst the ligation junctions in each lane, indicative of enrichment of higher fidelity triplet polymerases in the selection pool.
    Figure Legend Snippet: Clonal versus polyclonal activity. Type 1 and 2 RNAs, and the indicated polyclonal ribozyme pools late in the selection, were transcribed as selection constructs generated with Tri8AUAM (then annealed to primer A10). Their abilities to incorporate eight ppp AUA triplets then self-ligate in this selection construct context were compared (left, 0.1 μM each RNA, 2 μM ppp AUA, in −7˚C ice for 5 days; right, 0.2 μM each RNA, 2 μM ppp AUA and ppp AUG 3’d , in −7˚C ice for 9 days). Left: Despite being the most active type ( Figure 2b ), type two activity fell far short of the round 19 pool polyclonal activity. Right: The incorporation of wobble pairing 3’-deoxy ‘terminator’ triplet ppp AUG 3’d (see results section on fidelity, yielding a faster-migrating band than incorporation of the cognate ppp AUA triplet) was decreased sharply after round 19, as calculated by densitometry and averaging amongst the ligation junctions in each lane, indicative of enrichment of higher fidelity triplet polymerases in the selection pool.

    Techniques Used: Activity Assay, Selection, Construct, Generated, Ligation

    Fidelity of type 5 variants. Positional error rates and fidelities (determined as in Figure 8a ) for type 5 ribozyme variants; the overall fidelity was calculated as a geometric mean of positional errors at each triplet position (n and s.d. of this value shown for ribozymes assayed multiple times, see Figure 8—source data 1 ). Fidelity of the initial type 5 isolate (tethered to the assay templates) is modestly improved in the absence of type 1. However, with type 1, fidelity is improved when operating fully in trans (type5 s +1 ). Reselection and stabilisation of the ε domain yields a further fidelity improvement (t5 +1 ) but ε truncation (αβγδ +1 ) reverts the pattern of error tendencies along the triplet towards that of the starting core. See Supplementary file 1 for ribozyme sequences.
    Figure Legend Snippet: Fidelity of type 5 variants. Positional error rates and fidelities (determined as in Figure 8a ) for type 5 ribozyme variants; the overall fidelity was calculated as a geometric mean of positional errors at each triplet position (n and s.d. of this value shown for ribozymes assayed multiple times, see Figure 8—source data 1 ). Fidelity of the initial type 5 isolate (tethered to the assay templates) is modestly improved in the absence of type 1. However, with type 1, fidelity is improved when operating fully in trans (type5 s +1 ). Reselection and stabilisation of the ε domain yields a further fidelity improvement (t5 +1 ) but ε truncation (αβγδ +1 ) reverts the pattern of error tendencies along the triplet towards that of the starting core. See Supplementary file 1 for ribozyme sequences.

    Techniques Used:

    Substrate competition attenuates inhibitory ε + /ε - pairing during self-synthesis. ( A ) Longer oligonucleotide substrates are required to maximise synthesis of full-length ε + segment (0.5 µM Fε9 primer/Tε template/oligonucleotide substrate and 5 µM each triplet (left, omitted where equivalent oligonucleotide substrate was present), 13 days in −7˚C ice). Replacing two triplets with a preformed hexanucleotide substrate ( ppp UGAAUG) boosts full-length ε + product synthesis by t5 +1 (left panel), but not by the type 6 s+1 ribozyme - with a different accessory domain (see Figure 2—figure supplement 1 , sequence in Supplementary file 1 ) - which synthesizes ε + independent of this hexanucleotide (right panel, allowing some full-length ε + synthesis with only triplets). This likely reflects unfavourable competition between the t5 ribozyme’s own ε + domain and triplet substrates for complementary pairing to the ε - RNA used as template. Consistent with this hypothesis, increasing t5 +1 concentrations with this template is counterproductive (middle panel). For both ribozymes, using a preformed nonanucleotide substrate ppp UUUUUCAUG at the end of the template boosted full-length product over use of a ppp UUCAUG hexanucleotide + ppp UUU triplet, or just the three constituent triplets (S = 9 vs. 6/3 vs. 3/3/3); this is not due to the individual triplet sequences as t5 +1 can efficiently incorporate both ppp UUU and ppp UUC (see Figure 5b , Figure 6a ). ( B ) Doubling triplet concentrations to 10 µM can attenuate t5 +1 ’s requirement for the ppp UGAAUG hexanucleotide substrate during ε + synthesis. This suggests that at higher triplet concentrations they successfully compete with this part of t5 for ε - template hybridisation (0.5 µM Fε9 primer/Tε template/longer oligonucleotide substrates, 13 days in −7˚C ice). With a fourfold excess of primer/template duplex and substrates (right lane), the amount of full-length segment generated exceeds the ribozyme added, evidence of multiple turnover of full-length ε + product.
    Figure Legend Snippet: Substrate competition attenuates inhibitory ε + /ε - pairing during self-synthesis. ( A ) Longer oligonucleotide substrates are required to maximise synthesis of full-length ε + segment (0.5 µM Fε9 primer/Tε template/oligonucleotide substrate and 5 µM each triplet (left, omitted where equivalent oligonucleotide substrate was present), 13 days in −7˚C ice). Replacing two triplets with a preformed hexanucleotide substrate ( ppp UGAAUG) boosts full-length ε + product synthesis by t5 +1 (left panel), but not by the type 6 s+1 ribozyme - with a different accessory domain (see Figure 2—figure supplement 1 , sequence in Supplementary file 1 ) - which synthesizes ε + independent of this hexanucleotide (right panel, allowing some full-length ε + synthesis with only triplets). This likely reflects unfavourable competition between the t5 ribozyme’s own ε + domain and triplet substrates for complementary pairing to the ε - RNA used as template. Consistent with this hypothesis, increasing t5 +1 concentrations with this template is counterproductive (middle panel). For both ribozymes, using a preformed nonanucleotide substrate ppp UUUUUCAUG at the end of the template boosted full-length product over use of a ppp UUCAUG hexanucleotide + ppp UUU triplet, or just the three constituent triplets (S = 9 vs. 6/3 vs. 3/3/3); this is not due to the individual triplet sequences as t5 +1 can efficiently incorporate both ppp UUU and ppp UUC (see Figure 5b , Figure 6a ). ( B ) Doubling triplet concentrations to 10 µM can attenuate t5 +1 ’s requirement for the ppp UGAAUG hexanucleotide substrate during ε + synthesis. This suggests that at higher triplet concentrations they successfully compete with this part of t5 for ε - template hybridisation (0.5 µM Fε9 primer/Tε template/longer oligonucleotide substrates, 13 days in −7˚C ice). With a fourfold excess of primer/template duplex and substrates (right lane), the amount of full-length segment generated exceeds the ribozyme added, evidence of multiple turnover of full-length ε + product.

    Techniques Used: Sequencing, Hybridization, Generated

    Summary of ribozyme development in this work. Left, map of triplet polymerase selection and engineering. Ribozyme sequences are listed in Supplementary file 1 . Right, secondary structures of the central ribozymes in this work.
    Figure Legend Snippet: Summary of ribozyme development in this work. Left, map of triplet polymerase selection and engineering. Ribozyme sequences are listed in Supplementary file 1 . Right, secondary structures of the central ribozymes in this work.

    Techniques Used: Selection

    Ribozyme segment synthesis with mixed length substrate pools. δ + (left) or γ + (right) syntheses were performed (using 0.5 µM each of t5 +1 and the primer/templates above, 15 days in −7˚C ice) with their constitutive triplet sets alone (*, see Figure 6a ) or the random-sequence substrate mixes indicated. Introduction of dinucleotide and mononucleotide substrates ( ppp NN, ppp N i.e. NTPs) decreases full-length product band intensity but not ligations performed (here quantified whilst assuming each extension product was formed from the fewest substrates possible), with increasing numbers of extension products deviating from the starting triplet register. Dinucleotides and mononucleotides appear poor substrates in the absence of triplets.
    Figure Legend Snippet: Ribozyme segment synthesis with mixed length substrate pools. δ + (left) or γ + (right) syntheses were performed (using 0.5 µM each of t5 +1 and the primer/templates above, 15 days in −7˚C ice) with their constitutive triplet sets alone (*, see Figure 6a ) or the random-sequence substrate mixes indicated. Introduction of dinucleotide and mononucleotide substrates ( ppp NN, ppp N i.e. NTPs) decreases full-length product band intensity but not ligations performed (here quantified whilst assuming each extension product was formed from the fewest substrates possible), with increasing numbers of extension products deviating from the starting triplet register. Dinucleotides and mononucleotides appear poor substrates in the absence of triplets.

    Techniques Used: Sequencing

    Determination of residual fidelity phenotype when using minor groove-modified substrates. Top: framework for estimation of residual fidelity phenotype (ϕ). Shown are model logistic curves for incorporation of a triplet, for example GAU (at a fixed concentration, opposite 3’-AUC-5’), versus increasing concentrations of a mismatching triplet, for example GGU. A ribozyme ‘+’ with a fidelity function (e.g. t5 a , blue curve) will incorporate equal amounts of the matched and mismatched triplets at a different concentration of the mismatched triplet compared to a ribozyme ‘−’ without a fidelity function (e.g. αβγδ, red curve). x + and x − represent the lns of these concentrations for + and −; their separation (x + - x − ) is a proxy for the strength of the ‘+’ fidelity phenotype. We measured the relative incorporation (W) of triplet pairs by the two triplet polymerases at test concentrations (t) of mismatched triplet, chosen to maximise the difference in the ribozymes’ resulting fractional incorporations (marked by a blue dot vs. a red dot on respective curves). If a triplet modification ( * , right) interferes with mismatch discrimination by ‘+’, it would shift the ‘+’ relative incorporation curve (blue) towards that of ‘−’ (red), reducing the difference in fractional incorporation. Assuming curve steepness (k) remains constant, the residual phenotype (ϕ) for that modification is described by the new separation (x +* - x −* ) as a proportion of the original separation (x + - x − ); numerical values and calculations from the measurements described below are supplied in Figure 8—source data 2 . Middle: measurements of ratios of incorporation vs. misincorporation (W) for unmodified and modified triplet pairs (at the indicated test concentrations) by the t5 a +1 triplet polymerase compared to the ε fidelity domain-truncated αβγδ +1 (at 0.5 μM each). The expected triplet additions are in grey along the left of each gel; the average W from n independent experiments, calculated via densitometry of products containing slower-migrating G misincorporations, is shown below each lane along with the average ϕ. Primers (P), templates (T) and additional triplets were at 0.5/0.5/5 μM, incubated for three days in −7˚C ice. Left: presence of a 2-thiouracil (2SU) at the third position of the triplet abolishes the fidelity domain’s ability to discriminate against a second position wobble pair (P: A10, T: CCCMisGAU, +5 μM ppp CCC). Centre-left: the same third position modification (2SU) also abolished the fidelity domain’s preference for G misincorporation at the first position (P: Fγ7, T: TγAGU, +5 μM HO CUG). Centre-right: in contrast, a 2SU at the first position of the triplet exerted no clear influence upon third position wobble discrimination (P: A10, T: CCCMisUGA, +5 μM ppp CCC). Right: replacement of a 2’ hydroxyl group with a 2’ fluoro (2’F) at the first triplet position likewise had no effect upon third position mismatch discrimination (P: Fγ7, T: TγAGU, +5 μM HO CUG). Below: Measurement of fidelity phenotype in the presence or absence of a downstream triplet. Residual phenotypes indicate attenuation or abolishment of third position (left panel, P Fγ7, T TγAGU), second position (middle panel, P Fγ7, T TγGAU), and third position (right panel, P Fγ7, T TγAGU) fidelity effects from downstream triplet absence. No effects are seen (upon third position discrimination) from the presence or absence of a 5’ triphosphate on the downstream triplet (ϕ ppp ). In the absence of a triplet bound downstream on the template, the fidelity phenotype is severely compromised, suggesting this adjacent triplet:template duplex plays a critical role in positioning the fidelity domain relative to the incoming triplet.
    Figure Legend Snippet: Determination of residual fidelity phenotype when using minor groove-modified substrates. Top: framework for estimation of residual fidelity phenotype (ϕ). Shown are model logistic curves for incorporation of a triplet, for example GAU (at a fixed concentration, opposite 3’-AUC-5’), versus increasing concentrations of a mismatching triplet, for example GGU. A ribozyme ‘+’ with a fidelity function (e.g. t5 a , blue curve) will incorporate equal amounts of the matched and mismatched triplets at a different concentration of the mismatched triplet compared to a ribozyme ‘−’ without a fidelity function (e.g. αβγδ, red curve). x + and x − represent the lns of these concentrations for + and −; their separation (x + - x − ) is a proxy for the strength of the ‘+’ fidelity phenotype. We measured the relative incorporation (W) of triplet pairs by the two triplet polymerases at test concentrations (t) of mismatched triplet, chosen to maximise the difference in the ribozymes’ resulting fractional incorporations (marked by a blue dot vs. a red dot on respective curves). If a triplet modification ( * , right) interferes with mismatch discrimination by ‘+’, it would shift the ‘+’ relative incorporation curve (blue) towards that of ‘−’ (red), reducing the difference in fractional incorporation. Assuming curve steepness (k) remains constant, the residual phenotype (ϕ) for that modification is described by the new separation (x +* - x −* ) as a proportion of the original separation (x + - x − ); numerical values and calculations from the measurements described below are supplied in Figure 8—source data 2 . Middle: measurements of ratios of incorporation vs. misincorporation (W) for unmodified and modified triplet pairs (at the indicated test concentrations) by the t5 a +1 triplet polymerase compared to the ε fidelity domain-truncated αβγδ +1 (at 0.5 μM each). The expected triplet additions are in grey along the left of each gel; the average W from n independent experiments, calculated via densitometry of products containing slower-migrating G misincorporations, is shown below each lane along with the average ϕ. Primers (P), templates (T) and additional triplets were at 0.5/0.5/5 μM, incubated for three days in −7˚C ice. Left: presence of a 2-thiouracil (2SU) at the third position of the triplet abolishes the fidelity domain’s ability to discriminate against a second position wobble pair (P: A10, T: CCCMisGAU, +5 μM ppp CCC). Centre-left: the same third position modification (2SU) also abolished the fidelity domain’s preference for G misincorporation at the first position (P: Fγ7, T: TγAGU, +5 μM HO CUG). Centre-right: in contrast, a 2SU at the first position of the triplet exerted no clear influence upon third position wobble discrimination (P: A10, T: CCCMisUGA, +5 μM ppp CCC). Right: replacement of a 2’ hydroxyl group with a 2’ fluoro (2’F) at the first triplet position likewise had no effect upon third position mismatch discrimination (P: Fγ7, T: TγAGU, +5 μM HO CUG). Below: Measurement of fidelity phenotype in the presence or absence of a downstream triplet. Residual phenotypes indicate attenuation or abolishment of third position (left panel, P Fγ7, T TγAGU), second position (middle panel, P Fγ7, T TγGAU), and third position (right panel, P Fγ7, T TγAGU) fidelity effects from downstream triplet absence. No effects are seen (upon third position discrimination) from the presence or absence of a 5’ triphosphate on the downstream triplet (ϕ ppp ). In the absence of a triplet bound downstream on the template, the fidelity phenotype is severely compromised, suggesting this adjacent triplet:template duplex plays a critical role in positioning the fidelity domain relative to the incoming triplet.

    Techniques Used: Modification, Concentration Assay, Incubation, Countercurrent Chromatography

    Ribozyme catalytic domain self-synthesis and assembly. Top, conditions and yields for self-synthesis and assembly of the catalytic t5 domain (as αβ + and γδε + fragments, Figure 6b ) by t5 +1 . To obtain maximal amounts of fully synthesized and assembled t5 b ribozyme for activity testing ( Figure 6c ), we implemented t5 ‘+’ strand synthesis using sequence-specified triplet substrates for segment syntheses (as shown). The synthesis scale corresponds to the limiting component present. *: Unlike other segment ligation steps, the δε ligation was carried out using freeze-thaw cycling ( Mutschler et al., 2015 ) (2.5 hr at −30˚C, 21 hr at −7˚C, then 0.5 hr at 37˚C), which modestly improved yield. Below, scheme of t5 +1 -catalysed t5 synthesis and assembly reactions. PAGE separations of syntheses (xt) alongside purified reference segments and fragments (in bold) were stained with SYBR Gold to quantify boxed full-length products. These were excised for use in subsequent assembly steps (illustrated by dashed black arrows). Synthetic schemes of colour-coded segments are shown beside corresponding lanes, denoting ‘+’ strand synthesis (bold primer and dashes) on ‘–’ strand template. Fully synthesized segments are shown as bold lines. Assembly reactions involve use of fully synthesized segments as the final substrate in a primer extension reaction (α + β, γ + δε) or direct templated ligation of synthesized segments (δ + ε). The αβ + and γδε + fragments associate spontaneously to form an active triplet polymerase ribozyme ( Figure 6b ), tested for activity in Figure 6c . The synthesised segment sequences were derived from the t5 b variant of t5, which differs by one neutral signature mutation from t5 in the α segment, and by another neutral signature mutation from t5 a in the ε segment (mutations highlighted in Supplementary file 1 ); these neutral mutations, not present in the t5 and t5 a used to synthesise the segments (above), allowed verification of the synthetic origin of the product fragments by sequencing, which revealed the correct signature mutations in each fragment ruling out contamination of the synthesized ‘+’ strand products by the synthesizing (‘+’ strand) triplet polymerase ribozyme.
    Figure Legend Snippet: Ribozyme catalytic domain self-synthesis and assembly. Top, conditions and yields for self-synthesis and assembly of the catalytic t5 domain (as αβ + and γδε + fragments, Figure 6b ) by t5 +1 . To obtain maximal amounts of fully synthesized and assembled t5 b ribozyme for activity testing ( Figure 6c ), we implemented t5 ‘+’ strand synthesis using sequence-specified triplet substrates for segment syntheses (as shown). The synthesis scale corresponds to the limiting component present. *: Unlike other segment ligation steps, the δε ligation was carried out using freeze-thaw cycling ( Mutschler et al., 2015 ) (2.5 hr at −30˚C, 21 hr at −7˚C, then 0.5 hr at 37˚C), which modestly improved yield. Below, scheme of t5 +1 -catalysed t5 synthesis and assembly reactions. PAGE separations of syntheses (xt) alongside purified reference segments and fragments (in bold) were stained with SYBR Gold to quantify boxed full-length products. These were excised for use in subsequent assembly steps (illustrated by dashed black arrows). Synthetic schemes of colour-coded segments are shown beside corresponding lanes, denoting ‘+’ strand synthesis (bold primer and dashes) on ‘–’ strand template. Fully synthesized segments are shown as bold lines. Assembly reactions involve use of fully synthesized segments as the final substrate in a primer extension reaction (α + β, γ + δε) or direct templated ligation of synthesized segments (δ + ε). The αβ + and γδε + fragments associate spontaneously to form an active triplet polymerase ribozyme ( Figure 6b ), tested for activity in Figure 6c . The synthesised segment sequences were derived from the t5 b variant of t5, which differs by one neutral signature mutation from t5 in the α segment, and by another neutral signature mutation from t5 a in the ε segment (mutations highlighted in Supplementary file 1 ); these neutral mutations, not present in the t5 and t5 a used to synthesise the segments (above), allowed verification of the synthetic origin of the product fragments by sequencing, which revealed the correct signature mutations in each fragment ruling out contamination of the synthesized ‘+’ strand products by the synthesizing (‘+’ strand) triplet polymerase ribozyme.

    Techniques Used: Synthesized, Activity Assay, Sequencing, Ligation, Polyacrylamide Gel Electrophoresis, Purification, Staining, Derivative Assay, Variant Assay, Mutagenesis

    Parameters of type 1 activity enhancement. ( A ) PAGE of primer extension reactions comprising combinations of type 5 and type 1 variants (0.2 μM primer A10, template SR3, and type 5 variant, here annealed together and combined with 0.2 μM of separately-annealed type 1 variant after buffer addition; −7˚C in ice for 63 hr, 2 μM each ppp AUA, ppp AUG 3’d , ppp CGC). Full-length type 1 enhanced the activity of type 5 as did 5’ truncated type 1 (‘1’, Figure 3a ) without its duplex-interacting A-minor motif ( Shechner et al., 2009 ) and its ‘cap−’ sequence (originally present on some type 1 sequences in the selection). Replacing the type 5 ribozyme’s selected 5’ ‘cap+’ sequence with ‘cap-’ (type 5 cap- ) abolished its enhancement by type 1 RNA. ( B ) Stoichiometry of type 1 enhancement of type 3 activity (0.5 μM primer A10, template SR3, 2 μM each ppp AUA, ppp GUA 3’d , ppp CGC in −7˚C ice for 20 hr); quantification of average extension per primer in each lane is consistent with the 1:1 stoichiometry implied by type 1’s ~ 50% abundance in the round 21 selection pool.
    Figure Legend Snippet: Parameters of type 1 activity enhancement. ( A ) PAGE of primer extension reactions comprising combinations of type 5 and type 1 variants (0.2 μM primer A10, template SR3, and type 5 variant, here annealed together and combined with 0.2 μM of separately-annealed type 1 variant after buffer addition; −7˚C in ice for 63 hr, 2 μM each ppp AUA, ppp AUG 3’d , ppp CGC). Full-length type 1 enhanced the activity of type 5 as did 5’ truncated type 1 (‘1’, Figure 3a ) without its duplex-interacting A-minor motif ( Shechner et al., 2009 ) and its ‘cap−’ sequence (originally present on some type 1 sequences in the selection). Replacing the type 5 ribozyme’s selected 5’ ‘cap+’ sequence with ‘cap-’ (type 5 cap- ) abolished its enhancement by type 1 RNA. ( B ) Stoichiometry of type 1 enhancement of type 3 activity (0.5 μM primer A10, template SR3, 2 μM each ppp AUA, ppp GUA 3’d , ppp CGC in −7˚C ice for 20 hr); quantification of average extension per primer in each lane is consistent with the 1:1 stoichiometry implied by type 1’s ~ 50% abundance in the round 21 selection pool.

    Techniques Used: Activity Assay, Polyacrylamide Gel Electrophoresis, Variant Assay, Sequencing, Selection

    Ribozyme stabilisation attenuates inhibitory δ + /δ - pairing during self-synthesis. Left, δ + synthesis using 5 μM of each complementary triplet substrate (−hex) or 1 μM of ppp UGACAU (+hex) replacing the final two triplets (0.25 μM primer Fδ7/template HTδ, 0.5 μM each ribozyme, 6 days in −7˚C ice). Type 5 with the initially isolated ε domain (type 5 s ) needs hexamer substrate for δ + synthesis, but with the reselected ε domain (t5 a ) does not. See Supplementary file 1 for ribozyme sequences. Right, model of substrate/ribozyme competition for template binding. The region at the end of the δ segment exhibits irregular pairing in the initially isolated ε domain (above), and appears vulnerable to δ - template pairing during δ synthesis with triplet substrates. Reselection strengthened base-pairing in the corresponding ε domain stem (below), replacing a G-U wobble with a cognate G-C pair and a U.U mispair with a cognate U-A pair, stabilizing the δ - ε domain junction and potentially reducing competition with triplets during δ segment synthesis.
    Figure Legend Snippet: Ribozyme stabilisation attenuates inhibitory δ + /δ - pairing during self-synthesis. Left, δ + synthesis using 5 μM of each complementary triplet substrate (−hex) or 1 μM of ppp UGACAU (+hex) replacing the final two triplets (0.25 μM primer Fδ7/template HTδ, 0.5 μM each ribozyme, 6 days in −7˚C ice). Type 5 with the initially isolated ε domain (type 5 s ) needs hexamer substrate for δ + synthesis, but with the reselected ε domain (t5 a ) does not. See Supplementary file 1 for ribozyme sequences. Right, model of substrate/ribozyme competition for template binding. The region at the end of the δ segment exhibits irregular pairing in the initially isolated ε domain (above), and appears vulnerable to δ - template pairing during δ synthesis with triplet substrates. Reselection strengthened base-pairing in the corresponding ε domain stem (below), replacing a G-U wobble with a cognate G-C pair and a U.U mispair with a cognate U-A pair, stabilizing the δ - ε domain junction and potentially reducing competition with triplets during δ segment synthesis.

    Techniques Used: Isolation, Binding Assay

    7) Product Images from "hMSH2 forms specific mispair-binding complexes with hMSH3 and hMSH6"

    Article Title: hMSH2 forms specific mispair-binding complexes with hMSH3 and hMSH6

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    Tissue expression of hMSH6 mRNA. A human multiple-tissue Northern blot (CLONTECH) was probed with the 32 P-labeled complete cDNA clone of hMSH6 according to the manufacturer’s protocol. Colon refers to mucosal lining; p.b. leukocyte refers to peripheral blood leukocyte. The hMSH6 transcript is indicated by the arrow and corresponds to ≈4.5 kb. The amount of RNA loaded in each lane was adjusted to comparable levels as judged spectrophotometrically and by the levels of actin present (CLONTECH).
    Figure Legend Snippet: Tissue expression of hMSH6 mRNA. A human multiple-tissue Northern blot (CLONTECH) was probed with the 32 P-labeled complete cDNA clone of hMSH6 according to the manufacturer’s protocol. Colon refers to mucosal lining; p.b. leukocyte refers to peripheral blood leukocyte. The hMSH6 transcript is indicated by the arrow and corresponds to ≈4.5 kb. The amount of RNA loaded in each lane was adjusted to comparable levels as judged spectrophotometrically and by the levels of actin present (CLONTECH).

    Techniques Used: Expressing, Northern Blot, Labeling

    8) Product Images from "The RNA-binding complex ESCRT-II in Xenopus laevis eggs recognizes purine-rich sequences through its subunit, Vps25"

    Article Title: The RNA-binding complex ESCRT-II in Xenopus laevis eggs recognizes purine-rich sequences through its subunit, Vps25

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.003718

    Analysis of ESCRT-II/RNA binding in vitro . A , Coomassie Blue-stained gel of the recombinant Xenopus ( Xen ) and human ( Hu ) ESCRT-II complexes used in the in vitro RNA-binding assays. ΔMBD lacks the membrane-binding domains of human ESCRT-II. B–D , autoradiographs of UV–cross-linked in vitro binding reactions with: B , Xenopus ESCRT-II and 5′-end-labeled total egg RNA; C , Xenopus ESCRT-II and individual 5′-end-labeled in vitro transcribed RNAs that are under-represented in ESCRT-II immunoprecipitations; and D , full-length HuESCRT-II ( FL ) or HuESCRT-IIΔMBD (ΔMBD) and a body-labeled, in vitro transcribed GA-rich CLIP tag (a region of the ctr9 mRNA). B and C , a covalent intermediate of PNK and [γ- 32 P]ATP (used to radiolabel the RNA fragments) is indicated. D , Folch fraction liposomes were included in the binding reactions at the indicated concentrations. A fluorescent Western blotting ( WB ) of the same nitrocellulose membrane shown in the autoradiograph is shown as a loading control. The asterisk represents a nonspecific band. A–D , the expected migrations of the ESCRT-II subunits are indicated. E , quantification of the autoradiograph shown in D and two additional, independent replicates depicting the fraction of RNA bound by each ESCRT-II subunit at the indicated concentrations of Folch fraction liposomes relative to binding with no liposomes present. Error bars are S.E.
    Figure Legend Snippet: Analysis of ESCRT-II/RNA binding in vitro . A , Coomassie Blue-stained gel of the recombinant Xenopus ( Xen ) and human ( Hu ) ESCRT-II complexes used in the in vitro RNA-binding assays. ΔMBD lacks the membrane-binding domains of human ESCRT-II. B–D , autoradiographs of UV–cross-linked in vitro binding reactions with: B , Xenopus ESCRT-II and 5′-end-labeled total egg RNA; C , Xenopus ESCRT-II and individual 5′-end-labeled in vitro transcribed RNAs that are under-represented in ESCRT-II immunoprecipitations; and D , full-length HuESCRT-II ( FL ) or HuESCRT-IIΔMBD (ΔMBD) and a body-labeled, in vitro transcribed GA-rich CLIP tag (a region of the ctr9 mRNA). B and C , a covalent intermediate of PNK and [γ- 32 P]ATP (used to radiolabel the RNA fragments) is indicated. D , Folch fraction liposomes were included in the binding reactions at the indicated concentrations. A fluorescent Western blotting ( WB ) of the same nitrocellulose membrane shown in the autoradiograph is shown as a loading control. The asterisk represents a nonspecific band. A–D , the expected migrations of the ESCRT-II subunits are indicated. E , quantification of the autoradiograph shown in D and two additional, independent replicates depicting the fraction of RNA bound by each ESCRT-II subunit at the indicated concentrations of Folch fraction liposomes relative to binding with no liposomes present. Error bars are S.E.

    Techniques Used: RNA Binding Assay, In Vitro, Staining, Recombinant, Binding Assay, Labeling, Cross-linking Immunoprecipitation, Western Blot, Autoradiography

    9) Product Images from "Small RNA-seq: The RNA 5’-end adapter ligation problem and how to circumvent it"

    Article Title: Small RNA-seq: The RNA 5’-end adapter ligation problem and how to circumvent it

    Journal: Journal of Biological Methods

    doi: 10.14440/jbm.2019.269

    Monitoring Coligo-seq steps for Pol III transcription template Dcr3, and Illumina sequencing. A . Schematic representation of different species formed during library preparation. Asterisk, 5’- 32 P-end-label/ligation site; black dot, the single ribonucleotide in RT primer. B . Denaturing gel of in vitro transcription (IVT) and 3’ adapter ligation reaction products for coligo Dcr3 using Adapter3. C . Reverse transcription (RT) of gel purified 3’ adapter-ligated transcripts for cDNA synthesis using RTprimer2 followed by cDNA circularization and re-linearization with RNase A at the ribonucleotide site. L, linear cDNA first strand; C, circularization reaction product. D . PCR amplification of re-linearized cDNA template (V) using Primer2F and Primer2R2. Agarose gel. No-template control is indicated by ( - ). cDNA within the bracket was recovered for sequencing on Illumina MiSeq. E . Read length distribution of Dcr3 cDNA library after sequencing on Illumina MiSeq for 110 cycles followed by adapter trimming of each read. Horizontal bar below graph indicates the input RNA size range. Percentage of reads based on the total reads from the library. Compare peaks with transcript size in (B), lane 1. F . Whole-transcript read analysis for coligo Dcr3 cDNA library obtained after Illumina sequencing. The 10 most abundant reads are shown. Differently colored regions indicate different regions in predicted secondary structure of the coligo: larger loop (LL, green), smaller loop (SL, yellow), and stem including internal loops and bulges (cyan).
    Figure Legend Snippet: Monitoring Coligo-seq steps for Pol III transcription template Dcr3, and Illumina sequencing. A . Schematic representation of different species formed during library preparation. Asterisk, 5’- 32 P-end-label/ligation site; black dot, the single ribonucleotide in RT primer. B . Denaturing gel of in vitro transcription (IVT) and 3’ adapter ligation reaction products for coligo Dcr3 using Adapter3. C . Reverse transcription (RT) of gel purified 3’ adapter-ligated transcripts for cDNA synthesis using RTprimer2 followed by cDNA circularization and re-linearization with RNase A at the ribonucleotide site. L, linear cDNA first strand; C, circularization reaction product. D . PCR amplification of re-linearized cDNA template (V) using Primer2F and Primer2R2. Agarose gel. No-template control is indicated by ( - ). cDNA within the bracket was recovered for sequencing on Illumina MiSeq. E . Read length distribution of Dcr3 cDNA library after sequencing on Illumina MiSeq for 110 cycles followed by adapter trimming of each read. Horizontal bar below graph indicates the input RNA size range. Percentage of reads based on the total reads from the library. Compare peaks with transcript size in (B), lane 1. F . Whole-transcript read analysis for coligo Dcr3 cDNA library obtained after Illumina sequencing. The 10 most abundant reads are shown. Differently colored regions indicate different regions in predicted secondary structure of the coligo: larger loop (LL, green), smaller loop (SL, yellow), and stem including internal loops and bulges (cyan).

    Techniques Used: Sequencing, Ligation, In Vitro, Purification, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, cDNA Library Assay

    Coligo-seq variation for making a cDNA library from 5’ monophosphorylated small RNA.
    Figure Legend Snippet: Coligo-seq variation for making a cDNA library from 5’ monophosphorylated small RNA.

    Techniques Used: cDNA Library Assay

    Coligo-seq proof of concept trial. A . Diagram depicting the Coligo-seq library preparation steps, followed by sequencing (in this experiment, Sanger sequencing). Black dot denotes single ribonucleotide and p* denotes 5’- 32 P label. Roman numerals are used to indicate different species produced during library preparation steps and their corresponding gel species in panels (B) and (C). P1 and P2, Primer1F and Primer1R. B . 3’ adapter ligation of coligo 122 transcripts followed by reverse transcription to make cDNA first strand using RTprimer1. 5’ radiolabeled cDNA (III) was excised for circularization. C. cDNA first strand circularization using TS2126 RNA ligase 1 (Rnl1) and re-linearization using RNase A. Re-linearized cDNA product (V) was used as the template for PCR amplification. Concatamers might include linear or circular tandem dimers. D . PCR amplification of re-linearized cDNA using Primer1F and Primer1R. The band indicated by bracket was cut, gel eluted, cloned, and sequenced. Templates used for PCR are: no template control ( - ), RTprimer1 negative control (X), where primer binding sites diverge, and re-linearized cDNAs (species V). E . Sanger sequencing analysis of the coligo 122 transcripts (5 clones total). Transcription start site (Tss, arrow) and transcript 3’ end site (Stops) were previously identified via 5’ and 3’ RACE sequencing are indicated for comparison.
    Figure Legend Snippet: Coligo-seq proof of concept trial. A . Diagram depicting the Coligo-seq library preparation steps, followed by sequencing (in this experiment, Sanger sequencing). Black dot denotes single ribonucleotide and p* denotes 5’- 32 P label. Roman numerals are used to indicate different species produced during library preparation steps and their corresponding gel species in panels (B) and (C). P1 and P2, Primer1F and Primer1R. B . 3’ adapter ligation of coligo 122 transcripts followed by reverse transcription to make cDNA first strand using RTprimer1. 5’ radiolabeled cDNA (III) was excised for circularization. C. cDNA first strand circularization using TS2126 RNA ligase 1 (Rnl1) and re-linearization using RNase A. Re-linearized cDNA product (V) was used as the template for PCR amplification. Concatamers might include linear or circular tandem dimers. D . PCR amplification of re-linearized cDNA using Primer1F and Primer1R. The band indicated by bracket was cut, gel eluted, cloned, and sequenced. Templates used for PCR are: no template control ( - ), RTprimer1 negative control (X), where primer binding sites diverge, and re-linearized cDNAs (species V). E . Sanger sequencing analysis of the coligo 122 transcripts (5 clones total). Transcription start site (Tss, arrow) and transcript 3’ end site (Stops) were previously identified via 5’ and 3’ RACE sequencing are indicated for comparison.

    Techniques Used: Sequencing, Produced, Ligation, Polymerase Chain Reaction, Amplification, Clone Assay, Negative Control, Binding Assay

    Coligo-seq results for Pol III template Dcr3 mapped to Dcr3’s predicted secondary structure. The transcription start and stop sites were compiled from the cDNA Illumina sequencing results and mapped to the predicted secondary structure. Reads representing less than 1% of the total were excluded, so the absence of a green or red bar indicates
    Figure Legend Snippet: Coligo-seq results for Pol III template Dcr3 mapped to Dcr3’s predicted secondary structure. The transcription start and stop sites were compiled from the cDNA Illumina sequencing results and mapped to the predicted secondary structure. Reads representing less than 1% of the total were excluded, so the absence of a green or red bar indicates

    Techniques Used: Sequencing

    10) Product Images from "Fully 3D Printed Integrated Reactor Array for Point-of-Care Molecular Diagnostics"

    Article Title: Fully 3D Printed Integrated Reactor Array for Point-of-Care Molecular Diagnostics

    Journal: Biosensors & bioelectronics

    doi: 10.1016/j.bios.2018.03.009

    Colorimetric and fluorescence based detection for NAATs in 3D printed reactor array. A) Representative photographs of colorimetric LAMP assay for detection of N. meningitidis with 0, 50, 500 and 5000 CFU/reaction on the same chip, alongside LAMP fluorescence based image at given time interval. B) LAMP amplification curves for P. falciparum with 0, 0.1 1, 10, 100, 1000 pg per reaction. C) Calibration curve for P. falciparum as function of log target concentration, n=3. D) LAMP amplification curves for N. meningitidis with 0, 50, 500, 5000 CFU per reaction. E) Calibration curve for N. meningitidis as function of log target concentration, n=3. WarmStart ® LAMP master mix was used.
    Figure Legend Snippet: Colorimetric and fluorescence based detection for NAATs in 3D printed reactor array. A) Representative photographs of colorimetric LAMP assay for detection of N. meningitidis with 0, 50, 500 and 5000 CFU/reaction on the same chip, alongside LAMP fluorescence based image at given time interval. B) LAMP amplification curves for P. falciparum with 0, 0.1 1, 10, 100, 1000 pg per reaction. C) Calibration curve for P. falciparum as function of log target concentration, n=3. D) LAMP amplification curves for N. meningitidis with 0, 50, 500, 5000 CFU per reaction. E) Calibration curve for N. meningitidis as function of log target concentration, n=3. WarmStart ® LAMP master mix was used.

    Techniques Used: Fluorescence, Lamp Assay, Chromatin Immunoprecipitation, Amplification, Concentration Assay

    LAMP amplification curves of serial dilutions of P. falciparum gDNA in PBS samples with or without static coating in the 3D printed amplification reactors. A) no coating, B) BSA coating, C) PEG coating and D) PVA coating (n=3). Optigene ® LAMP Isothermal Master Mix was used. Note: rxn = reaction
    Figure Legend Snippet: LAMP amplification curves of serial dilutions of P. falciparum gDNA in PBS samples with or without static coating in the 3D printed amplification reactors. A) no coating, B) BSA coating, C) PEG coating and D) PVA coating (n=3). Optigene ® LAMP Isothermal Master Mix was used. Note: rxn = reaction

    Techniques Used: Amplification

    11) Product Images from "Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA"

    Article Title: Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA

    Journal: RNA

    doi: 10.1261/rna.2242610

    Optimization of RNA 3′-end adapter ligation. ( A ) Temperature optimization. Synthetic ssRNA oligonucleotides with either 2′-hydroxyl (OH) or 2′- O -methyl ( O -Me) 3′-ends were ligated to pre-adenylated DNA adapters (Linker) at different temperatures for either 2 or 18 h with 200 units of T4 Rnl2tr or without enzyme (−; input control). Ligation products were resolved and visualized by SYBR Gold staining. Ligation efficiency at varying temperatures is graphically represented as the mean ± SEM of four independent experiments. ( B ) Polyethylene glycol (PEG) as a ligation enhancer. Ligations were performed in the presence of varying concentrations of polyethylene glycol 8000 (PEG). Final concentrations in the reaction were 6.25%, 12.5%, and 25% (w/v). Ligation reactions were incubated for either 2 h or 18 h at 22°C or 16°C as indicated using 200 units of T4 Rnl2tr. (−) Indicates the absence of ligase. Ligation efficiency at varying concentrations of PEG 8000 is graphically represented as the mean ± SEM of three independent experiments. ( C ) Enzyme concentration. Ligations were performed using increasing amounts truncated T4 Rnl2tr (0, 10, 50, 100, 200, 500, 1000 units) in a reaction buffer containing 25% PEG 8000 (w/v) for 2 h at room temperature. Ligation efficiency using increasing amounts of enzyme are graphically represented as the mean ± SEM of three independent experiments.
    Figure Legend Snippet: Optimization of RNA 3′-end adapter ligation. ( A ) Temperature optimization. Synthetic ssRNA oligonucleotides with either 2′-hydroxyl (OH) or 2′- O -methyl ( O -Me) 3′-ends were ligated to pre-adenylated DNA adapters (Linker) at different temperatures for either 2 or 18 h with 200 units of T4 Rnl2tr or without enzyme (−; input control). Ligation products were resolved and visualized by SYBR Gold staining. Ligation efficiency at varying temperatures is graphically represented as the mean ± SEM of four independent experiments. ( B ) Polyethylene glycol (PEG) as a ligation enhancer. Ligations were performed in the presence of varying concentrations of polyethylene glycol 8000 (PEG). Final concentrations in the reaction were 6.25%, 12.5%, and 25% (w/v). Ligation reactions were incubated for either 2 h or 18 h at 22°C or 16°C as indicated using 200 units of T4 Rnl2tr. (−) Indicates the absence of ligase. Ligation efficiency at varying concentrations of PEG 8000 is graphically represented as the mean ± SEM of three independent experiments. ( C ) Enzyme concentration. Ligations were performed using increasing amounts truncated T4 Rnl2tr (0, 10, 50, 100, 200, 500, 1000 units) in a reaction buffer containing 25% PEG 8000 (w/v) for 2 h at room temperature. Ligation efficiency using increasing amounts of enzyme are graphically represented as the mean ± SEM of three independent experiments.

    Techniques Used: Ligation, Staining, Incubation, Concentration Assay

    RNA 3′-end attachment. ( A ) Comparison of optimized T4 Rnl2tr ligation to published ligation conditions. Synthetic ssRNA oligonucleotides with either 2′-hydroxyl (OH) or 2′- O -methyl ( O -Me) 3′-ends were ligated to pre-adenylated DNA adapter (AppLinker) using T4 Rnl2tr or T4 Rnl1 under different ligation conditions (conditions 1, 2, 3; detailed in Materials and Methods). Ligation products were resolved and visualized by SYBR Gold staining. ( B ) Quantification of ligation efficiency. Percent ligation refers to the amount of input RNA converted to ligated species as measured by densitometry. Data points represent the mean ± SEM; n = 3 experimental replicates.
    Figure Legend Snippet: RNA 3′-end attachment. ( A ) Comparison of optimized T4 Rnl2tr ligation to published ligation conditions. Synthetic ssRNA oligonucleotides with either 2′-hydroxyl (OH) or 2′- O -methyl ( O -Me) 3′-ends were ligated to pre-adenylated DNA adapter (AppLinker) using T4 Rnl2tr or T4 Rnl1 under different ligation conditions (conditions 1, 2, 3; detailed in Materials and Methods). Ligation products were resolved and visualized by SYBR Gold staining. ( B ) Quantification of ligation efficiency. Percent ligation refers to the amount of input RNA converted to ligated species as measured by densitometry. Data points represent the mean ± SEM; n = 3 experimental replicates.

    Techniques Used: Ligation, Staining

    RNA 3′-end adapter ligation bias against 2′- O -methylated small RNA 3′-ends. Synthetic ssRNA oligonucleotides with either 2′-hydroxyl (OH) or 2′- O -methyl ( O -Me) 3′-ends and different 3′-terminal nucleotides (A, C, G, or U) were ligated to a pre-adenylated DNA adapter (AppLinker) using either T4 Rnl2tr or T4 Rnl1. Ligation products were resolved and visualized by SYBR Gold staining. Percent ligation refers to the relative amount of input RNA converted to ligated species as measured by densitometry. Data points represent the mean ± SEM; n = 3 experimental replicates.
    Figure Legend Snippet: RNA 3′-end adapter ligation bias against 2′- O -methylated small RNA 3′-ends. Synthetic ssRNA oligonucleotides with either 2′-hydroxyl (OH) or 2′- O -methyl ( O -Me) 3′-ends and different 3′-terminal nucleotides (A, C, G, or U) were ligated to a pre-adenylated DNA adapter (AppLinker) using either T4 Rnl2tr or T4 Rnl1. Ligation products were resolved and visualized by SYBR Gold staining. Percent ligation refers to the relative amount of input RNA converted to ligated species as measured by densitometry. Data points represent the mean ± SEM; n = 3 experimental replicates.

    Techniques Used: Ligation, Methylation, Staining

    12) Product Images from "High-Throughput Single-Cell Labeling (Hi-SCL) for RNA-Seq Using Drop-Based Microfluidics"

    Article Title: High-Throughput Single-Cell Labeling (Hi-SCL) for RNA-Seq Using Drop-Based Microfluidics

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0116328

    Experimental workflow of Hi-SCL RNA-Seq. A) To prepare the cell-labels, a barcode-library emulsion is produced using a microfluidic device consisting of 96 drop-makers each of which precisely fit into the wells of one quarter of a 384 well-plate. All of the drop-makers share a common oil inlet and a common outlet. The microfluidic device and the well-plate are placed in a pressurized chamber, causing the content of each of the 96 wells to flow into the respective drop-maker. This creates millions of drops containing a high concentration of a single one of the 96 barcodes stored in the well plate. The drops from all wells are mixed to form the library. B) Using a microfluidic drop-maker, cells are co-encapsulated with lysis buffer at a filling number of one per 10 drops. C) After incubating the emulsion of cell-bearing drops, it is re-injected, together with the emulsion containing the barcode-drops, into a second microfluidic device where each cell-bearing drop is paired with one barcode-drop. As the two adjacent drops flow through a microfluidic junction they enter a region containing an electric field induced by the electrodes. This induces coalescence of the drops; simultaneously, buffer containing RT enzyme is injected into the merged drops. D right) Each barcode is a single DNA strand designed with a polyT at the 5’ end, followed by a unique barcode sequence and a fixed priming region (PRIMER 1). After merging the barcode drop with a drop containing a lysed cell, the 5’ end of each barcode hybridizes to a cellular mRNA. Subsequently, an RT reaction produces a cDNA strand that is tagged with a cell-specific barcode. D middle) After the RT reaction is complete, the emulsion is broken and a single stranded DNA is ligated to all cDNA strands. The tagged strands are amplified using the two priming regions, one on each end. D left) Gel electrophoresis of 4 samples of 100 cells after amplification.
    Figure Legend Snippet: Experimental workflow of Hi-SCL RNA-Seq. A) To prepare the cell-labels, a barcode-library emulsion is produced using a microfluidic device consisting of 96 drop-makers each of which precisely fit into the wells of one quarter of a 384 well-plate. All of the drop-makers share a common oil inlet and a common outlet. The microfluidic device and the well-plate are placed in a pressurized chamber, causing the content of each of the 96 wells to flow into the respective drop-maker. This creates millions of drops containing a high concentration of a single one of the 96 barcodes stored in the well plate. The drops from all wells are mixed to form the library. B) Using a microfluidic drop-maker, cells are co-encapsulated with lysis buffer at a filling number of one per 10 drops. C) After incubating the emulsion of cell-bearing drops, it is re-injected, together with the emulsion containing the barcode-drops, into a second microfluidic device where each cell-bearing drop is paired with one barcode-drop. As the two adjacent drops flow through a microfluidic junction they enter a region containing an electric field induced by the electrodes. This induces coalescence of the drops; simultaneously, buffer containing RT enzyme is injected into the merged drops. D right) Each barcode is a single DNA strand designed with a polyT at the 5’ end, followed by a unique barcode sequence and a fixed priming region (PRIMER 1). After merging the barcode drop with a drop containing a lysed cell, the 5’ end of each barcode hybridizes to a cellular mRNA. Subsequently, an RT reaction produces a cDNA strand that is tagged with a cell-specific barcode. D middle) After the RT reaction is complete, the emulsion is broken and a single stranded DNA is ligated to all cDNA strands. The tagged strands are amplified using the two priming regions, one on each end. D left) Gel electrophoresis of 4 samples of 100 cells after amplification.

    Techniques Used: RNA Sequencing Assay, Produced, Flow Cytometry, Concentration Assay, Lysis, Injection, Sequencing, Amplification, Nucleic Acid Electrophoresis

    13) Product Images from "Dis3l2-Mediated Decay Is a Quality Control Pathway for Noncoding RNAs"

    Article Title: Dis3l2-Mediated Decay Is a Quality Control Pathway for Noncoding RNAs

    Journal: Cell reports

    doi: 10.1016/j.celrep.2016.07.025

    Deep sequencing analysis of the 3′-end of Rmrp RNA (A) Schematic protocol for Rmrp cRACE. See the method section for details. (B) The distribution and length of U-tails in input and FLAG-mutant Dis3l2-IP RNAs. Note the 10–12 nucleotide-long peak in the U-tail length in the Dis3l2-bound Rmrp reads. (C) Analysis of Rmrp 3′ end revealed that U-tails are mainly added to transcripts with 2 additional nucleotides at their 3′ compared to annotated gene. (D) In input samples, majority of Rmrp species are terminated 1 nucleotide before the annotated transcript (highlighted in red), while the U-tailed Rmrp reads in IP samples always end 2 nucleotides downstream.
    Figure Legend Snippet: Deep sequencing analysis of the 3′-end of Rmrp RNA (A) Schematic protocol for Rmrp cRACE. See the method section for details. (B) The distribution and length of U-tails in input and FLAG-mutant Dis3l2-IP RNAs. Note the 10–12 nucleotide-long peak in the U-tail length in the Dis3l2-bound Rmrp reads. (C) Analysis of Rmrp 3′ end revealed that U-tails are mainly added to transcripts with 2 additional nucleotides at their 3′ compared to annotated gene. (D) In input samples, majority of Rmrp species are terminated 1 nucleotide before the annotated transcript (highlighted in red), while the U-tailed Rmrp reads in IP samples always end 2 nucleotides downstream.

    Techniques Used: Sequencing, Mutagenesis

    Systematic identification of Dis3l2-associated RNAs in mouse ESCs (A) Western blot analysis of Dis3l2 expression in CRISPR-Cas9 generated knockout ESCs; WT, wild type; Het, heterozygous; KO, knockout. (B) Western blot analysis of FLAG-tagged Dis3l2 in input and IP samples. Arrow points to the FLAG-mutant Dis3l2 expression in knockout ESCs (Inputs) and after anti-FLAG immunoprecipitation (IPs). Asterisk shows an unspecific band, representing equal loading. Empty pFLAG-CMV2 vector was used as mock. (C) Scatter plotting of enriched transcripts in FLAG-mutant Dis3l2 IP compared to Mock IP. Among the most enriched transcripts are Rmrp, Rpph1, and the two Rpph1 pseudogenes, Rprl2 and Rprl3, all highlighted in red. (D) Representative sequencing track of Rmrp and Rpph1 in inputs and IPs. Read numbers range from 0–3500. (E) qRT-PCR analysis of FLAG-immunoprecipitated RNAs (n=6). Note that Malat1 was not enriched in FLAG-mutant Dis3l2 IP. To measure relative enrichment of each gene, expressions in IP samples were first normalized to Gapdh level and then to the respective input; ns, not significant. F) Anti-FLAG immunoblotting of Dis3l2 knockout cellular lysates after overexpression of WT, mutant Dis3l2, or mock transfected cells as well as related immunoprecipitation. (G) .
    Figure Legend Snippet: Systematic identification of Dis3l2-associated RNAs in mouse ESCs (A) Western blot analysis of Dis3l2 expression in CRISPR-Cas9 generated knockout ESCs; WT, wild type; Het, heterozygous; KO, knockout. (B) Western blot analysis of FLAG-tagged Dis3l2 in input and IP samples. Arrow points to the FLAG-mutant Dis3l2 expression in knockout ESCs (Inputs) and after anti-FLAG immunoprecipitation (IPs). Asterisk shows an unspecific band, representing equal loading. Empty pFLAG-CMV2 vector was used as mock. (C) Scatter plotting of enriched transcripts in FLAG-mutant Dis3l2 IP compared to Mock IP. Among the most enriched transcripts are Rmrp, Rpph1, and the two Rpph1 pseudogenes, Rprl2 and Rprl3, all highlighted in red. (D) Representative sequencing track of Rmrp and Rpph1 in inputs and IPs. Read numbers range from 0–3500. (E) qRT-PCR analysis of FLAG-immunoprecipitated RNAs (n=6). Note that Malat1 was not enriched in FLAG-mutant Dis3l2 IP. To measure relative enrichment of each gene, expressions in IP samples were first normalized to Gapdh level and then to the respective input; ns, not significant. F) Anti-FLAG immunoblotting of Dis3l2 knockout cellular lysates after overexpression of WT, mutant Dis3l2, or mock transfected cells as well as related immunoprecipitation. (G) .

    Techniques Used: Western Blot, Expressing, CRISPR, Generated, Knock-Out, Mutagenesis, Immunoprecipitation, Plasmid Preparation, Sequencing, Quantitative RT-PCR, Over Expression, Transfection

    14) Product Images from "Ribozyme-catalysed RNA synthesis using triplet building blocks"

    Article Title: Ribozyme-catalysed RNA synthesis using triplet building blocks

    Journal: eLife

    doi: 10.7554/eLife.35255

    Ribozyme catalytic domain self-synthesis and assembly. Top, conditions and yields for self-synthesis and assembly of the catalytic t5 domain (as αβ + and γδε + fragments, Figure 6b ) by t5 +1 . To obtain maximal amounts of fully synthesized and assembled t5 b ribozyme for activity testing ( Figure 6c ), we implemented t5 ‘+’ strand synthesis using sequence-specified triplet substrates for segment syntheses (as shown). The synthesis scale corresponds to the limiting component present. *: Unlike other segment ligation steps, the δε ligation was carried out using freeze-thaw cycling ( Mutschler et al., 2015 ) (2.5 hr at −30˚C, 21 hr at −7˚C, then 0.5 hr at 37˚C), which modestly improved yield. Below, scheme of t5 +1 -catalysed t5 synthesis and assembly reactions. PAGE separations of syntheses (xt) alongside purified reference segments and fragments (in bold) were stained with SYBR Gold to quantify boxed full-length products. These were excised for use in subsequent assembly steps (illustrated by dashed black arrows). Synthetic schemes of colour-coded segments are shown beside corresponding lanes, denoting ‘+’ strand synthesis (bold primer and dashes) on ‘–’ strand template. Fully synthesized segments are shown as bold lines. Assembly reactions involve use of fully synthesized segments as the final substrate in a primer extension reaction (α + β, γ + δε) or direct templated ligation of synthesized segments (δ + ε). The αβ + and γδε + fragments associate spontaneously to form an active triplet polymerase ribozyme ( Figure 6b ), tested for activity in Figure 6c . The synthesised segment sequences were derived from the t5 b variant of t5, which differs by one neutral signature mutation from t5 in the α segment, and by another neutral signature mutation from t5 a in the ε segment (mutations highlighted in Supplementary file 1 ); these neutral mutations, not present in the t5 and t5 a used to synthesise the segments (above), allowed verification of the synthetic origin of the product fragments by sequencing, which revealed the correct signature mutations in each fragment ruling out contamination of the synthesized ‘+’ strand products by the synthesizing (‘+’ strand) triplet polymerase ribozyme.
    Figure Legend Snippet: Ribozyme catalytic domain self-synthesis and assembly. Top, conditions and yields for self-synthesis and assembly of the catalytic t5 domain (as αβ + and γδε + fragments, Figure 6b ) by t5 +1 . To obtain maximal amounts of fully synthesized and assembled t5 b ribozyme for activity testing ( Figure 6c ), we implemented t5 ‘+’ strand synthesis using sequence-specified triplet substrates for segment syntheses (as shown). The synthesis scale corresponds to the limiting component present. *: Unlike other segment ligation steps, the δε ligation was carried out using freeze-thaw cycling ( Mutschler et al., 2015 ) (2.5 hr at −30˚C, 21 hr at −7˚C, then 0.5 hr at 37˚C), which modestly improved yield. Below, scheme of t5 +1 -catalysed t5 synthesis and assembly reactions. PAGE separations of syntheses (xt) alongside purified reference segments and fragments (in bold) were stained with SYBR Gold to quantify boxed full-length products. These were excised for use in subsequent assembly steps (illustrated by dashed black arrows). Synthetic schemes of colour-coded segments are shown beside corresponding lanes, denoting ‘+’ strand synthesis (bold primer and dashes) on ‘–’ strand template. Fully synthesized segments are shown as bold lines. Assembly reactions involve use of fully synthesized segments as the final substrate in a primer extension reaction (α + β, γ + δε) or direct templated ligation of synthesized segments (δ + ε). The αβ + and γδε + fragments associate spontaneously to form an active triplet polymerase ribozyme ( Figure 6b ), tested for activity in Figure 6c . The synthesised segment sequences were derived from the t5 b variant of t5, which differs by one neutral signature mutation from t5 in the α segment, and by another neutral signature mutation from t5 a in the ε segment (mutations highlighted in Supplementary file 1 ); these neutral mutations, not present in the t5 and t5 a used to synthesise the segments (above), allowed verification of the synthetic origin of the product fragments by sequencing, which revealed the correct signature mutations in each fragment ruling out contamination of the synthesized ‘+’ strand products by the synthesizing (‘+’ strand) triplet polymerase ribozyme.

    Techniques Used: Synthesized, Activity Assay, Sequencing, Ligation, Polyacrylamide Gel Electrophoresis, Purification, Staining, Derivative Assay, Variant Assay, Mutagenesis

    15) Product Images from "NEAT1 Scaffolds RNA Binding Proteins and the Microprocessor to Globally Enhance Pri-miRNA Processing"

    Article Title: NEAT1 Scaffolds RNA Binding Proteins and the Microprocessor to Globally Enhance Pri-miRNA Processing

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.3455

    Genome-wide analysis of NONO-PSF-RNA interactions ( a ) Immunoprecipitated NONO-PSF crosslinked to RNA. The complex was treated with two different concentrations of MNase (1:1,000 or 1:50,000 dilution); RNA in the complex was 32 p-labeled with T4 polynucleotide kinase. Proteins and RNA were visualized by Western blotting (left) and autoradiography (right). Indicated bands (right) were individually isolated for CLIP-seq library construction. ( b ) Venn Diagram showing overlapped pri-miRNAs bound by NONO and PSF in HeLa cells. ( c ) Footprint of NONO and PSF on pri-miRNAs. ( d ) Representative NONO and PSF binding tracks on the pri-miR-17–92a transcript. ( e ) The binding profiles of NONO and PSF on NEAT1 in comparison with the published DGCR8 CLIP-seq signals 44 . Y-axis in d and e shows CLIP-seq read density in each case. The region encoding for miR-612 is indicated at bottom. Uncropped images of Western blots and autoradiography in a are shown in Supplementary Data Set 1 .
    Figure Legend Snippet: Genome-wide analysis of NONO-PSF-RNA interactions ( a ) Immunoprecipitated NONO-PSF crosslinked to RNA. The complex was treated with two different concentrations of MNase (1:1,000 or 1:50,000 dilution); RNA in the complex was 32 p-labeled with T4 polynucleotide kinase. Proteins and RNA were visualized by Western blotting (left) and autoradiography (right). Indicated bands (right) were individually isolated for CLIP-seq library construction. ( b ) Venn Diagram showing overlapped pri-miRNAs bound by NONO and PSF in HeLa cells. ( c ) Footprint of NONO and PSF on pri-miRNAs. ( d ) Representative NONO and PSF binding tracks on the pri-miR-17–92a transcript. ( e ) The binding profiles of NONO and PSF on NEAT1 in comparison with the published DGCR8 CLIP-seq signals 44 . Y-axis in d and e shows CLIP-seq read density in each case. The region encoding for miR-612 is indicated at bottom. Uncropped images of Western blots and autoradiography in a are shown in Supplementary Data Set 1 .

    Techniques Used: Genome Wide, Immunoprecipitation, Labeling, Western Blot, Autoradiography, Isolation, Cross-linking Immunoprecipitation, Binding Assay

    16) Product Images from "3′READS+, a sensitive and accurate method for 3′ end sequencing of polyadenylated RNA"

    Article Title: 3′READS+, a sensitive and accurate method for 3′ end sequencing of polyadenylated RNA

    Journal: RNA

    doi: 10.1261/rna.057075.116

    Optimization of 5′ and 3′ adapter ligation steps. ( A ) Ligation protocols tested. In protocol A, ligation with 3′ and 5′ adapters was performed sequentially in the same tube. The 5′ adapter is an RNA oligo with hydroxyl
    Figure Legend Snippet: Optimization of 5′ and 3′ adapter ligation steps. ( A ) Ligation protocols tested. In protocol A, ligation with 3′ and 5′ adapters was performed sequentially in the same tube. The 5′ adapter is an RNA oligo with hydroxyl

    Techniques Used: Ligation

    3′READS+. ( A ) The 3′READS+ protocol incorporating optimized RNase H digestion and ligation steps. AAA n , poly(A) tail; A n , shortened poly(A) tail. 5′ adapter, 3′ adapter, random sequences in the adapters (3× N's),
    Figure Legend Snippet: 3′READS+. ( A ) The 3′READS+ protocol incorporating optimized RNase H digestion and ligation steps. AAA n , poly(A) tail; A n , shortened poly(A) tail. 5′ adapter, 3′ adapter, random sequences in the adapters (3× N's),

    Techniques Used: Ligation

    Optimization of 5′ and 3′ adapter ligation steps. ( A ) Ligation protocols tested. In protocol A, ligation with 3′ and 5′ adapters was performed sequentially in the same tube. The 5′ adapter is an RNA oligo with hydroxyl
    Figure Legend Snippet: Optimization of 5′ and 3′ adapter ligation steps. ( A ) Ligation protocols tested. In protocol A, ligation with 3′ and 5′ adapters was performed sequentially in the same tube. The 5′ adapter is an RNA oligo with hydroxyl

    Techniques Used: Ligation

    3′READS+. ( A ) The 3′READS+ protocol incorporating optimized RNase H digestion and ligation steps. AAA n , poly(A) tail; A n , shortened poly(A) tail. 5′ adapter, 3′ adapter, random sequences in the adapters (3× N's),
    Figure Legend Snippet: 3′READS+. ( A ) The 3′READS+ protocol incorporating optimized RNase H digestion and ligation steps. AAA n , poly(A) tail; A n , shortened poly(A) tail. 5′ adapter, 3′ adapter, random sequences in the adapters (3× N's),

    Techniques Used: Ligation

    17) Product Images from "Fully 3D Printed Integrated Reactor Array for Point-of-Care Molecular Diagnostics"

    Article Title: Fully 3D Printed Integrated Reactor Array for Point-of-Care Molecular Diagnostics

    Journal: Biosensors & bioelectronics

    doi: 10.1016/j.bios.2018.03.009

    LAMP amplification curves of serial dilutions of P. falciparum gDNA in PBS samples with or without static coating in the 3D printed amplification reactors. A) no coating, B) BSA coating, C) PEG coating and D) PVA coating (n=3). Optigene ® LAMP Isothermal Master Mix was used. Note: rxn = reaction
    Figure Legend Snippet: LAMP amplification curves of serial dilutions of P. falciparum gDNA in PBS samples with or without static coating in the 3D printed amplification reactors. A) no coating, B) BSA coating, C) PEG coating and D) PVA coating (n=3). Optigene ® LAMP Isothermal Master Mix was used. Note: rxn = reaction

    Techniques Used: Amplification

    18) Product Images from "A ligation-based single-stranded library preparation method to analyze cell-free DNA and synthetic oligos"

    Article Title: A ligation-based single-stranded library preparation method to analyze cell-free DNA and synthetic oligos

    Journal: BMC Genomics

    doi: 10.1186/s12864-019-6355-0

    Standard NGS metrics for merged reads from SRSLY and NEBNext Ultra II libraries from healthy human cfDNA extracts H-69 and H-81. Unless otherwise stated, all libraries for each method were combined by cfDNA extract prior to analysis and filtered for PCR duplicates and a quality score equal to or greater than q20. ( a ) Insert distribution plots for cfDNA extracts H-69 and H-81, respectively. ( b ) Fold coverage by base percent across the human genome ( hg19 ) for SRSLY and NEBNext by cfDNA extract. Combined libraries were subsampled to similar read depth prior to fold coverage calculations. Subsampled depth was set at 295 M reads, the limit of sequenced reads for SRSLY-H-81. ( c ) Preseq complexity estimate for SRSLY and NEBNext by cfDNA extract. Three libraries of equivalent sequencing depth per method were combined to estimate complexity, since more libraries were made via SRSLY than NEBNext. Files containing the PCR duplicate reads were used to facilitate complexity estimates ( d ) Normalized coverage as a function of GC content over 100 bp sliding scale across the human genome for SRSLY and NEBNext by cfDNA extract. Green histogram represents the human genome GC across the 100 bp sliding window. ( e ) Normalized, log-transformed base composition at each position of read termini starting 2 bp upstream and extending to 34 bp downstream of read start site for combined cfDNA extracts for SRSLY and NEBNext. All reads regardless of insert length considered
    Figure Legend Snippet: Standard NGS metrics for merged reads from SRSLY and NEBNext Ultra II libraries from healthy human cfDNA extracts H-69 and H-81. Unless otherwise stated, all libraries for each method were combined by cfDNA extract prior to analysis and filtered for PCR duplicates and a quality score equal to or greater than q20. ( a ) Insert distribution plots for cfDNA extracts H-69 and H-81, respectively. ( b ) Fold coverage by base percent across the human genome ( hg19 ) for SRSLY and NEBNext by cfDNA extract. Combined libraries were subsampled to similar read depth prior to fold coverage calculations. Subsampled depth was set at 295 M reads, the limit of sequenced reads for SRSLY-H-81. ( c ) Preseq complexity estimate for SRSLY and NEBNext by cfDNA extract. Three libraries of equivalent sequencing depth per method were combined to estimate complexity, since more libraries were made via SRSLY than NEBNext. Files containing the PCR duplicate reads were used to facilitate complexity estimates ( d ) Normalized coverage as a function of GC content over 100 bp sliding scale across the human genome for SRSLY and NEBNext by cfDNA extract. Green histogram represents the human genome GC across the 100 bp sliding window. ( e ) Normalized, log-transformed base composition at each position of read termini starting 2 bp upstream and extending to 34 bp downstream of read start site for combined cfDNA extracts for SRSLY and NEBNext. All reads regardless of insert length considered

    Techniques Used: Next-Generation Sequencing, Polymerase Chain Reaction, Sequencing, Transformation Assay

    cfDNA analysis. ( a ) Normalized genomic dinucleotide frequencies as a function of read length for SRSLY data for three discrete fragment lengths including 100 bp ± the read mapped coordinates. Read midpoint is centered at 0. Negative numbers denote genomic regions upstream (5-prime) of the midpoint and positive numbers denote genomic regions downstream (3-prime) of the midpoint. Input data is from the combined H-69 and H-81 SRSLY datasets. ( b ) Same as (a) except for NEBNext data. ( c ) Normalized genomic dinucleotide frequency as a function of read length for SRSLY data for the termini of three discrete fragment lengths including a 9 bp region into the read (positive numbers) and 10 bp outside the read (negative numbers). Read start and end coordinates are centered on 0. Input data is from the combined H-69 and H-81 SRSLY datasets. ( d ) Same as (c) except for NEBNext data. ( e ) Normalized WPS values (120 bp window; 120–180 bp fragments) for SRSLY data compared to sample CH01 [ 16 ] at the same pericentromeric locus on chromosome 12 used to initially showcase WPS. ( f ) Average normalized WPS score within ±1 kb of annotated CTCF binding sites for long fragment length binned data (120 bp window, 120–180 bp fragments) and short fragment length binned data (16 bp window, 35–80 bp fragments) for SRSLY data compared to sample CH01 [ 16 ]
    Figure Legend Snippet: cfDNA analysis. ( a ) Normalized genomic dinucleotide frequencies as a function of read length for SRSLY data for three discrete fragment lengths including 100 bp ± the read mapped coordinates. Read midpoint is centered at 0. Negative numbers denote genomic regions upstream (5-prime) of the midpoint and positive numbers denote genomic regions downstream (3-prime) of the midpoint. Input data is from the combined H-69 and H-81 SRSLY datasets. ( b ) Same as (a) except for NEBNext data. ( c ) Normalized genomic dinucleotide frequency as a function of read length for SRSLY data for the termini of three discrete fragment lengths including a 9 bp region into the read (positive numbers) and 10 bp outside the read (negative numbers). Read start and end coordinates are centered on 0. Input data is from the combined H-69 and H-81 SRSLY datasets. ( d ) Same as (c) except for NEBNext data. ( e ) Normalized WPS values (120 bp window; 120–180 bp fragments) for SRSLY data compared to sample CH01 [ 16 ] at the same pericentromeric locus on chromosome 12 used to initially showcase WPS. ( f ) Average normalized WPS score within ±1 kb of annotated CTCF binding sites for long fragment length binned data (120 bp window, 120–180 bp fragments) and short fragment length binned data (16 bp window, 35–80 bp fragments) for SRSLY data compared to sample CH01 [ 16 ]

    Techniques Used: Binding Assay

    19) Product Images from "Controlled phage therapy by photothermal ablation of specific bacterial species using gold nanorods targeted by chimeric phages"

    Article Title: Controlled phage therapy by photothermal ablation of specific bacterial species using gold nanorods targeted by chimeric phages

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1913234117

    Treatment of P. aeruginosa biofilm grown on MDCKII cells using M13-g3p(Pf1)–AuNRs and biocompatibility of phage–AuNRs with MDCKII cells. ( A ) PrestoBlue cell viability assay results for M13-g3p(Pf1)–AuNR treatment and irradiation of MDCKII cells grown alone (blue), M13-g3p(Pf1)–AuNR treatment and irradiation of P. aeruginosa biofilm grown on MDCKII cells (red), and M13-g3p(Pf1)–AuNR treatment and irradiation of P. aeruginosa biofilm alone (grown without MDCKII cells; black), over time during irradiation. The control (magenta) is MilliQ water alone. In this assay, the PrestoBlue reagent is modified by the reducing environment of live cells and becomes fluorescent; both MDCKII and P. aeruginosa cells contribute to PrestoBlue fluorescence. MDCKII cells (blue) are largely viable upon M13-g3p(Pf1)–AuNR treatment and irradiation, while P. aeruginosa cells (black) are killed by M13-g3p(Pf1)–AuNR treatment and irradiation over the time course shown. As expected, the PrestoBlue fluorescence of the biofilm grown on MDCKII cells during M13-g3p(Pf1)–AuNR treatment and irradiation (red) is roughly equal to the sum of the fluorescence of MDCKII cells treated and irradiated alone (blue) plus the fluorescence of biofilm cells treated and irradiated alone (black). After 6 min of treatment and irradiation, the fluorescence of the biofilm grown on MDCKII cells (red) appears to be similar to that of MDCKII cells alone (treated and irradiated; blue), consistent with selective killing of P. aeruginosa . ( B ) Biocompatibility of phage–AuNRs was measured by PrestoBlue cell viability assay. M13KE–AuNRs or M13KE phages at different concentrations were incubated with MDCKII cells for 48 h without irradiation. The control is MDCKII cells alone (without M13KE–AuNRs or M13KE). Cell viability percentages were calculated by normalizing fluorescence intensity by the control fluorescence. The concentration of the bioconjugates and phages are given in units of micromolar concentration (1 μM ∼ 6 × 10 14 phage particles per mL). Error bars represent 1 SD calculated from triplicates.
    Figure Legend Snippet: Treatment of P. aeruginosa biofilm grown on MDCKII cells using M13-g3p(Pf1)–AuNRs and biocompatibility of phage–AuNRs with MDCKII cells. ( A ) PrestoBlue cell viability assay results for M13-g3p(Pf1)–AuNR treatment and irradiation of MDCKII cells grown alone (blue), M13-g3p(Pf1)–AuNR treatment and irradiation of P. aeruginosa biofilm grown on MDCKII cells (red), and M13-g3p(Pf1)–AuNR treatment and irradiation of P. aeruginosa biofilm alone (grown without MDCKII cells; black), over time during irradiation. The control (magenta) is MilliQ water alone. In this assay, the PrestoBlue reagent is modified by the reducing environment of live cells and becomes fluorescent; both MDCKII and P. aeruginosa cells contribute to PrestoBlue fluorescence. MDCKII cells (blue) are largely viable upon M13-g3p(Pf1)–AuNR treatment and irradiation, while P. aeruginosa cells (black) are killed by M13-g3p(Pf1)–AuNR treatment and irradiation over the time course shown. As expected, the PrestoBlue fluorescence of the biofilm grown on MDCKII cells during M13-g3p(Pf1)–AuNR treatment and irradiation (red) is roughly equal to the sum of the fluorescence of MDCKII cells treated and irradiated alone (blue) plus the fluorescence of biofilm cells treated and irradiated alone (black). After 6 min of treatment and irradiation, the fluorescence of the biofilm grown on MDCKII cells (red) appears to be similar to that of MDCKII cells alone (treated and irradiated; blue), consistent with selective killing of P. aeruginosa . ( B ) Biocompatibility of phage–AuNRs was measured by PrestoBlue cell viability assay. M13KE–AuNRs or M13KE phages at different concentrations were incubated with MDCKII cells for 48 h without irradiation. The control is MDCKII cells alone (without M13KE–AuNRs or M13KE). Cell viability percentages were calculated by normalizing fluorescence intensity by the control fluorescence. The concentration of the bioconjugates and phages are given in units of micromolar concentration (1 μM ∼ 6 × 10 14 phage particles per mL). Error bars represent 1 SD calculated from triplicates.

    Techniques Used: Viability Assay, Irradiation, Modification, Fluorescence, Incubation, Concentration Assay

    Interaction between M13KE, AuNR, and E. coli cells. TEM image of M13KE–AuNR ( A ) illustrates conjugation of filamentous phage and AuNRs. When E. coli cells were mixed with M13KE and HOOC-PEG–AuNR (nonconjugated), no aggregation or localization of AuNRs to the cells was seen ( B ), but HS-PEG-COOH-modified M13KE–AuNR bioconjugates attached to E. coli cells did result in visible aggregation of AuNRs on the cell surface ( C ). Aggregation at one end of the bacterium (in C ) presumably occurs near the position of the F pilus; stimulation of retraction by phage attachment may cause accumulation at the root of the pilus ( 57 , 58 ).
    Figure Legend Snippet: Interaction between M13KE, AuNR, and E. coli cells. TEM image of M13KE–AuNR ( A ) illustrates conjugation of filamentous phage and AuNRs. When E. coli cells were mixed with M13KE and HOOC-PEG–AuNR (nonconjugated), no aggregation or localization of AuNRs to the cells was seen ( B ), but HS-PEG-COOH-modified M13KE–AuNR bioconjugates attached to E. coli cells did result in visible aggregation of AuNRs on the cell surface ( C ). Aggregation at one end of the bacterium (in C ) presumably occurs near the position of the F pilus; stimulation of retraction by phage attachment may cause accumulation at the root of the pilus ( 57 , 58 ).

    Techniques Used: Transmission Electron Microscopy, Conjugation Assay, Modification

    20) Product Images from "T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis"

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1169

    Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Clone Assay

    Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Plasmid Preparation, Clone Assay, Transformation Assay

    21) Product Images from "Cellular reagents for diagnostics and synthetic biology"

    Article Title: Cellular reagents for diagnostics and synthetic biology

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0201681

    PCR and Gibson assembly using cellular reagents. (a) Schematic depicting cellular PCR followed by cellular Gibson assembly for constructing new plasmids. Bacteria harboring target plasmids are mixed with polymerase-expressing cellular reagents and PCR is initiated by adding appropriate primers, buffer, and dNTP. The resulting PCR products are incubated with cellular reagents expressing Gibson assembly enzymes–Taq DNA polymerase, Taq DNA ligase, and T5 exonuclease–to assemble the new construct. (b) Cellular PCR amplification of vector and insert fragments directly from E . coli bacteria bearing target DNA plasmids using 2 x 10 7 cells of Phusion cellular reagents. Assembly parts include: (i) “pATetO 6XHis full length” vector for two part assembly with Kan r cassette bearing appropriate overlapping ends, and (ii) “pUC19 Fragments 1 and 2” for three part assembly with Kan r cassette whose ends overlap with pUC19 vector fragments. (c) Gibson assembly of agarose gel purified and unpurified cellular PCR products using pure or cellular Gibson assembly reagents. In “negative control” samples the PCR products were incubated in Gibson reaction buffer without pure or cellular Gibson enzymes. “pATetO 6XHis + Kan r ”represents a two part Gibson assembly while “Puc19 Fragment 1 + pUC19 Fragment 2 + Kan r ” represents a three-part Gibson assembly. Representative number of clones recovered in each case in the presence of both ampicillin and kanamycin are reported.
    Figure Legend Snippet: PCR and Gibson assembly using cellular reagents. (a) Schematic depicting cellular PCR followed by cellular Gibson assembly for constructing new plasmids. Bacteria harboring target plasmids are mixed with polymerase-expressing cellular reagents and PCR is initiated by adding appropriate primers, buffer, and dNTP. The resulting PCR products are incubated with cellular reagents expressing Gibson assembly enzymes–Taq DNA polymerase, Taq DNA ligase, and T5 exonuclease–to assemble the new construct. (b) Cellular PCR amplification of vector and insert fragments directly from E . coli bacteria bearing target DNA plasmids using 2 x 10 7 cells of Phusion cellular reagents. Assembly parts include: (i) “pATetO 6XHis full length” vector for two part assembly with Kan r cassette bearing appropriate overlapping ends, and (ii) “pUC19 Fragments 1 and 2” for three part assembly with Kan r cassette whose ends overlap with pUC19 vector fragments. (c) Gibson assembly of agarose gel purified and unpurified cellular PCR products using pure or cellular Gibson assembly reagents. In “negative control” samples the PCR products were incubated in Gibson reaction buffer without pure or cellular Gibson enzymes. “pATetO 6XHis + Kan r ”represents a two part Gibson assembly while “Puc19 Fragment 1 + pUC19 Fragment 2 + Kan r ” represents a three-part Gibson assembly. Representative number of clones recovered in each case in the presence of both ampicillin and kanamycin are reported.

    Techniques Used: Polymerase Chain Reaction, Expressing, Incubation, Construct, Amplification, Plasmid Preparation, Agarose Gel Electrophoresis, Purification, Clone Assay

    22) Product Images from "Integrated analysis of directly captured microRNA targets reveals the impact of microRNAs on mammalian transcriptome"

    Article Title: Integrated analysis of directly captured microRNA targets reveals the impact of microRNAs on mammalian transcriptome

    Journal: bioRxiv

    doi: 10.1101/672469

    Comparison of performance between CLEAR-CLIP captured and TargetScan predicted targets. a , Overlap between miR-200 CLEAR-CLIP genes with a 7mer or 8mer and TargetScan predictions for miR-200s. b , Log2 fold change in gene expression upon induction of the miR-200b cluster is shown for CLEAR-CLIP genes with a 7mer or 8mer and TargetScan predicted conserved sites as compared to genes without a miR-200 high confidence site and not predicted as conserved by TargetScan. c , Log2 fold change in gene expression upon induction of the miR-200b cluster is shown for genes only in CLEAR-CLIP, only predicted by TargetScan or in CLEAR-CLIP and TargetScan as compared to genes without a miR-200 high confidence site and not predicted by TargetScan. d , A portion of the Brd4 3’UTR is shown with miR-200 CLEAR-CLIP reads (top track), miR-200 seed sites (middle track) and TargetScan sites (bottom track) indicated. e , A portion of the Ammecr1l 3’UTR is shown with miR-200 CLEAR-CLIP reads (top track), miR-200 seed sites (middle track) and TargetScan sites (bottom track) indicated. f , A portion of the Tnrc6a 3’UTR is shown with miR-200 CLEAR-CLIP reads (top track) and miR-200 seed sites (bottom track) indicated. g , Overlap between miR-205 CLEAR-CLIP genes with a 7mer or 8mer and TargetScan predicted conserved sites for miR-205. h , Log2 fold change in gene expression upon induction of miR-205 is shown for CLEAR-CLIP genes with a 7mer or 8mer and TargetScan predicted sites as compared to genes without a miR-205 high confidence site and not predicted as conserved by TargetScan. i , Log2 fold change in gene expression upon induction of miR-205 is shown for genes only in CLEAR-CLIP, only predicted by TargetScan or in CLEAR-CLIP and TargetScan as compared to genes without a miR-205 high confidence site and not predicted by TargetScan. j , Overlap between all miRNA:mRNA CLEAR-CLIP interactions with a 7mer or 8mer and all conserved TargetScan predictions. For all CDF plots the number of genes is shown in parenthesis and p-values were calculated using the Kolmogorov–Smirnov test.
    Figure Legend Snippet: Comparison of performance between CLEAR-CLIP captured and TargetScan predicted targets. a , Overlap between miR-200 CLEAR-CLIP genes with a 7mer or 8mer and TargetScan predictions for miR-200s. b , Log2 fold change in gene expression upon induction of the miR-200b cluster is shown for CLEAR-CLIP genes with a 7mer or 8mer and TargetScan predicted conserved sites as compared to genes without a miR-200 high confidence site and not predicted as conserved by TargetScan. c , Log2 fold change in gene expression upon induction of the miR-200b cluster is shown for genes only in CLEAR-CLIP, only predicted by TargetScan or in CLEAR-CLIP and TargetScan as compared to genes without a miR-200 high confidence site and not predicted by TargetScan. d , A portion of the Brd4 3’UTR is shown with miR-200 CLEAR-CLIP reads (top track), miR-200 seed sites (middle track) and TargetScan sites (bottom track) indicated. e , A portion of the Ammecr1l 3’UTR is shown with miR-200 CLEAR-CLIP reads (top track), miR-200 seed sites (middle track) and TargetScan sites (bottom track) indicated. f , A portion of the Tnrc6a 3’UTR is shown with miR-200 CLEAR-CLIP reads (top track) and miR-200 seed sites (bottom track) indicated. g , Overlap between miR-205 CLEAR-CLIP genes with a 7mer or 8mer and TargetScan predicted conserved sites for miR-205. h , Log2 fold change in gene expression upon induction of miR-205 is shown for CLEAR-CLIP genes with a 7mer or 8mer and TargetScan predicted sites as compared to genes without a miR-205 high confidence site and not predicted as conserved by TargetScan. i , Log2 fold change in gene expression upon induction of miR-205 is shown for genes only in CLEAR-CLIP, only predicted by TargetScan or in CLEAR-CLIP and TargetScan as compared to genes without a miR-205 high confidence site and not predicted by TargetScan. j , Overlap between all miRNA:mRNA CLEAR-CLIP interactions with a 7mer or 8mer and all conserved TargetScan predictions. For all CDF plots the number of genes is shown in parenthesis and p-values were calculated using the Kolmogorov–Smirnov test.

    Techniques Used: Cross-linking Immunoprecipitation, Expressing

    23) Product Images from "Arm-specific cleavage and mutation during reverse transcription of 2΄,5΄-branched RNA by Moloney murine leukemia virus reverse transcriptase"

    Article Title: Arm-specific cleavage and mutation during reverse transcription of 2΄,5΄-branched RNA by Moloney murine leukemia virus reverse transcriptase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx073

    Scheme of the splinted-ligation method in bRNA construction. In this method, a 2΄-5΄ linked ribo-guanosine (G)-nucleoside in an RNA strand containing the 5΄-segment and 2΄-arm (precursor 1) is transformed into a branchpoint nucleotide by ligation to an RNA strand representing the 3΄-arm (precursor 2). To do so, the two precursors are hybridized partially to a complementary RNA bridge. In this way, the 5΄-phosphate of precursor 2 is brought close to the free 3΄-hydroxyl of the 2΄-5΄ linked nucleoside of precursor 1. The two oligonucleotides are then joined by T4 RNA Ligase 2. Red, blue, and pink symbols ‘w’ represent RNA; the black line represents DNA. The 2΄-5΄ linked ribo-G-nucleoside in precursor 1 at nucleotide (nt) position 37 is highlighted. Nucleic acids downstream of a 2΄-5΄ linkage are plotted vertically in linear and branched oligonucleotides.
    Figure Legend Snippet: Scheme of the splinted-ligation method in bRNA construction. In this method, a 2΄-5΄ linked ribo-guanosine (G)-nucleoside in an RNA strand containing the 5΄-segment and 2΄-arm (precursor 1) is transformed into a branchpoint nucleotide by ligation to an RNA strand representing the 3΄-arm (precursor 2). To do so, the two precursors are hybridized partially to a complementary RNA bridge. In this way, the 5΄-phosphate of precursor 2 is brought close to the free 3΄-hydroxyl of the 2΄-5΄ linked nucleoside of precursor 1. The two oligonucleotides are then joined by T4 RNA Ligase 2. Red, blue, and pink symbols ‘w’ represent RNA; the black line represents DNA. The 2΄-5΄ linked ribo-G-nucleoside in precursor 1 at nucleotide (nt) position 37 is highlighted. Nucleic acids downstream of a 2΄-5΄ linkage are plotted vertically in linear and branched oligonucleotides.

    Techniques Used: Ligation, Transformation Assay

    24) Product Images from "End-bridging is required for pol μ to efficiently promote repair of noncomplementary ends by nonhomologous end joining"

    Article Title: End-bridging is required for pol μ to efficiently promote repair of noncomplementary ends by nonhomologous end joining

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn164

    Factors required for joining of noncomplementary ends. ( A ) A diagram of the standard 280 bp substrate labeled internally with 32 P (asterisk), and possessing 3′ TT overhangs. Arrows indicate direction of synthesis by pol μ after alignment of ends by core NHEJ factors Ku and XL. ( B ) All reactions used 5 nM DNA substrate as illustrated in (A), and products analyzed after 5 min reactions. 25 nM Ku and 50 nM XL were added as indicated (+). Polymerase μ or λ was added at 25 nM or 250 nM (10×). S, substrate; P, concatamer ligation products. ( C ) Reactions performed as in B except ddNTPs substituted for dNTPs, and synthesis at one end analyzed by denaturing PAGE as described in methods. S, substrate; P, +1 synthesis product.
    Figure Legend Snippet: Factors required for joining of noncomplementary ends. ( A ) A diagram of the standard 280 bp substrate labeled internally with 32 P (asterisk), and possessing 3′ TT overhangs. Arrows indicate direction of synthesis by pol μ after alignment of ends by core NHEJ factors Ku and XL. ( B ) All reactions used 5 nM DNA substrate as illustrated in (A), and products analyzed after 5 min reactions. 25 nM Ku and 50 nM XL were added as indicated (+). Polymerase μ or λ was added at 25 nM or 250 nM (10×). S, substrate; P, concatamer ligation products. ( C ) Reactions performed as in B except ddNTPs substituted for dNTPs, and synthesis at one end analyzed by denaturing PAGE as described in methods. S, substrate; P, +1 synthesis product.

    Techniques Used: Labeling, Non-Homologous End Joining, Ligation, Polyacrylamide Gel Electrophoresis

    25) Product Images from "End-bridging is required for pol μ to efficiently promote repair of noncomplementary ends by nonhomologous end joining"

    Article Title: End-bridging is required for pol μ to efficiently promote repair of noncomplementary ends by nonhomologous end joining

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn164

    Factors required for joining of noncomplementary ends. ( A ) A diagram of the standard 280 bp substrate labeled internally with 32 P (asterisk), and possessing 3′ TT overhangs. Arrows indicate direction of synthesis by pol μ after alignment of ends by core NHEJ factors Ku and XL. ( B ) All reactions used 5 nM DNA substrate as illustrated in (A), and products analyzed after 5 min reactions. 25 nM Ku and 50 nM XL were added as indicated (+). Polymerase μ or λ was added at 25 nM or 250 nM (10×). S, substrate; P, concatamer ligation products. ( C ) Reactions performed as in B except ddNTPs substituted for dNTPs, and synthesis at one end analyzed by denaturing PAGE as described in methods. S, substrate; P, +1 synthesis product.
    Figure Legend Snippet: Factors required for joining of noncomplementary ends. ( A ) A diagram of the standard 280 bp substrate labeled internally with 32 P (asterisk), and possessing 3′ TT overhangs. Arrows indicate direction of synthesis by pol μ after alignment of ends by core NHEJ factors Ku and XL. ( B ) All reactions used 5 nM DNA substrate as illustrated in (A), and products analyzed after 5 min reactions. 25 nM Ku and 50 nM XL were added as indicated (+). Polymerase μ or λ was added at 25 nM or 250 nM (10×). S, substrate; P, concatamer ligation products. ( C ) Reactions performed as in B except ddNTPs substituted for dNTPs, and synthesis at one end analyzed by denaturing PAGE as described in methods. S, substrate; P, +1 synthesis product.

    Techniques Used: Labeling, Non-Homologous End Joining, Ligation, Polyacrylamide Gel Electrophoresis

    26) Product Images from "Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells"

    Article Title: Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0158581

    Optimizations for second strand synthesis. (A) Schematic of the second strand synthesis procedure. Synthetic 5’ phosphorylated ODNs containing the lesion of interest are annealed to phagemid single-stranded DNA, complimentary strands are synthesised by T4 DNA polymerase, and ligated by T4 DNA ligase. (B) Second strand synthesis of HRAS construct using ssDNA purified by silica spin columns or anion-exchange columns. ssDNA purified by anion-exchange column produces high yields of covalently closed product. (C) Schematic of the alkaline gel analysis of the construct nicks positions. Double-digest of pcDNA3.1(+)-HRAS with SmaI and NdeI produces two fragments (labelled 1 and 2). If the synthetic ODN that becomes part of the transcribed strand is not ligated, the transcribed strand fragment 2 produces two smaller fragments (3 and 4). (D) Alkaline gel analysis of HRAS constructs. Negative control HRAS WT T5 exonuclease (T5 exo) treated, covalently closed construct produces only two bands and positive control Fpg nicked HRAS 8-oxoG constructs, treated and not treated with T5 exonuclease, produce the expected four bands. The anion-exchange purified HRAS WT construct produces only two bands, indicating the nicks following second strand synthesis occur at random positions.
    Figure Legend Snippet: Optimizations for second strand synthesis. (A) Schematic of the second strand synthesis procedure. Synthetic 5’ phosphorylated ODNs containing the lesion of interest are annealed to phagemid single-stranded DNA, complimentary strands are synthesised by T4 DNA polymerase, and ligated by T4 DNA ligase. (B) Second strand synthesis of HRAS construct using ssDNA purified by silica spin columns or anion-exchange columns. ssDNA purified by anion-exchange column produces high yields of covalently closed product. (C) Schematic of the alkaline gel analysis of the construct nicks positions. Double-digest of pcDNA3.1(+)-HRAS with SmaI and NdeI produces two fragments (labelled 1 and 2). If the synthetic ODN that becomes part of the transcribed strand is not ligated, the transcribed strand fragment 2 produces two smaller fragments (3 and 4). (D) Alkaline gel analysis of HRAS constructs. Negative control HRAS WT T5 exonuclease (T5 exo) treated, covalently closed construct produces only two bands and positive control Fpg nicked HRAS 8-oxoG constructs, treated and not treated with T5 exonuclease, produce the expected four bands. The anion-exchange purified HRAS WT construct produces only two bands, indicating the nicks following second strand synthesis occur at random positions.

    Techniques Used: Construct, Purification, Negative Control, Positive Control

    27) Product Images from "RNA-dependent chromatin association of transcription elongation factors and Pol II CTD kinases"

    Article Title: RNA-dependent chromatin association of transcription elongation factors and Pol II CTD kinases

    Journal: eLife

    doi: 10.7554/eLife.25637

    Confirmatory information on PAR-CLIP experiments. ( A ) Smoothed, raw PAR-CLIP signals (as measured by the number of PAR-CLIP U-to-C transitions per U site) over a set of 2532 selected mRNAs were aligned at their 5′-end (TSS), scaled to a common length, then averaged (Materials and methods). The color code shows the PAR-CLIP signal relative to the maximum PAR-CLIP signal of all profiles (dark blue). Since PAR-CLIP signals of Set1 and Spt5 were much higher than those of other EFs, Set1 and Spt5 occupancies were divided by a factor of 1.5 and 3, respectively, for visualization purposes. ( B ) Replicate measurements show very high correlations. Comparison of PAR-CLIP replicate measurements for Ctk2, Ctr9, Spt6 and Set1. Smoothed, averaged PAR-CLIP profiles and scatterplots comparing the number of observed crosslinking sites per transcript for all mRNAs shown in ( A ) using Pearson correlation are shown. ( C ) Confirmation that Paf1C interacts with RNA through its subunits Rtf1, Ctr9 and Cdc73. (top) SDS PAGE analysis of RNA binding to Paf1C subunits after IP (Coomassie stain). Paf1C subunits can be individually pulled down with IgG beads against the C-terminal TAP tag of each of the five Paf1C subunits. (bottom) SDS-PAGE analysis of radioactively labeled RNA that was co-precipitated with Paf1C subunits. Only subunits Rtf1, Ctr9 and Cdc73 show detectable co-precipitation with RNA, showing that PAR-CLIP signals are subunit-specific. DOI: http://dx.doi.org/10.7554/eLife.25637.004
    Figure Legend Snippet: Confirmatory information on PAR-CLIP experiments. ( A ) Smoothed, raw PAR-CLIP signals (as measured by the number of PAR-CLIP U-to-C transitions per U site) over a set of 2532 selected mRNAs were aligned at their 5′-end (TSS), scaled to a common length, then averaged (Materials and methods). The color code shows the PAR-CLIP signal relative to the maximum PAR-CLIP signal of all profiles (dark blue). Since PAR-CLIP signals of Set1 and Spt5 were much higher than those of other EFs, Set1 and Spt5 occupancies were divided by a factor of 1.5 and 3, respectively, for visualization purposes. ( B ) Replicate measurements show very high correlations. Comparison of PAR-CLIP replicate measurements for Ctk2, Ctr9, Spt6 and Set1. Smoothed, averaged PAR-CLIP profiles and scatterplots comparing the number of observed crosslinking sites per transcript for all mRNAs shown in ( A ) using Pearson correlation are shown. ( C ) Confirmation that Paf1C interacts with RNA through its subunits Rtf1, Ctr9 and Cdc73. (top) SDS PAGE analysis of RNA binding to Paf1C subunits after IP (Coomassie stain). Paf1C subunits can be individually pulled down with IgG beads against the C-terminal TAP tag of each of the five Paf1C subunits. (bottom) SDS-PAGE analysis of radioactively labeled RNA that was co-precipitated with Paf1C subunits. Only subunits Rtf1, Ctr9 and Cdc73 show detectable co-precipitation with RNA, showing that PAR-CLIP signals are subunit-specific. DOI: http://dx.doi.org/10.7554/eLife.25637.004

    Techniques Used: Cross-linking Immunoprecipitation, SDS Page, RNA Binding Assay, Staining, Labeling

    Normalization of PAR-CLIP data shown for two representative EFs, Ctk2 (top) and Spt5 (bottom), at mRNAs (left) versus CUTs (right). Smoothed, raw and normalized PAR-CLIP signals as shown in Figure 2D but averaged over mRNAs (left) and CUTs (right). After normalization, average mRNA and CUT profiles were rescaled together, setting the maximum occupancy to one and the minimum occupancy to 0. This shows that after Pol II PAR-CLIP normalization Spt5 signals are equally high at mRNAs and CUTs, while no RNA normalization leads to less Spt5 signal at CUTs (due to less transcription of CUTs) and total RNA normalization leads to increased Spt5 levels at CUTs (due to decreased detection of unstable transcripts). Since Spt5 binds Pol II immediately downstream of initiation, differences in Spt5 RNA binding between mRNAs and CUTs would not be expected, arguing that Pol II normalization helps to correctly interpret the data. DOI: http://dx.doi.org/10.7554/eLife.25637.006
    Figure Legend Snippet: Normalization of PAR-CLIP data shown for two representative EFs, Ctk2 (top) and Spt5 (bottom), at mRNAs (left) versus CUTs (right). Smoothed, raw and normalized PAR-CLIP signals as shown in Figure 2D but averaged over mRNAs (left) and CUTs (right). After normalization, average mRNA and CUT profiles were rescaled together, setting the maximum occupancy to one and the minimum occupancy to 0. This shows that after Pol II PAR-CLIP normalization Spt5 signals are equally high at mRNAs and CUTs, while no RNA normalization leads to less Spt5 signal at CUTs (due to less transcription of CUTs) and total RNA normalization leads to increased Spt5 levels at CUTs (due to decreased detection of unstable transcripts). Since Spt5 binds Pol II immediately downstream of initiation, differences in Spt5 RNA binding between mRNAs and CUTs would not be expected, arguing that Pol II normalization helps to correctly interpret the data. DOI: http://dx.doi.org/10.7554/eLife.25637.006

    Techniques Used: Cross-linking Immunoprecipitation, RNA Binding Assay

    Non-averaged elongation factor RNA occupancies over mRNAs and introns. ( A ) Transcript-wise Pol II normalized elongation factor PAR-CLIP occupancies. Smoothed occupancy profiles derived from PAR-CLIP data for a set of 2532 selected mRNAs. Transcripts were sorted by length and aligned at their 5′-end (transcription start site, TSS). Plots for Ctk2 and Spt5 are shown in Figure 2C . ( B ) Smoothed Pol II normalized PAR-CLIP occupancy profiles over all introns. Each line represents an intron, and introns were sorted by length and aligned at their 5′ splice site (5′SS). Only introns of lengths between 150 and 650 nt are shown. DOI: http://dx.doi.org/10.7554/eLife.25637.008
    Figure Legend Snippet: Non-averaged elongation factor RNA occupancies over mRNAs and introns. ( A ) Transcript-wise Pol II normalized elongation factor PAR-CLIP occupancies. Smoothed occupancy profiles derived from PAR-CLIP data for a set of 2532 selected mRNAs. Transcripts were sorted by length and aligned at their 5′-end (transcription start site, TSS). Plots for Ctk2 and Spt5 are shown in Figure 2C . ( B ) Smoothed Pol II normalized PAR-CLIP occupancy profiles over all introns. Each line represents an intron, and introns were sorted by length and aligned at their 5′ splice site (5′SS). Only introns of lengths between 150 and 650 nt are shown. DOI: http://dx.doi.org/10.7554/eLife.25637.008

    Techniques Used: Cross-linking Immunoprecipitation, Derivative Assay

    Asymmetric distribution of EFs at coding and non-coding transcripts of similar length. ( A ) PAR-CLIP occupancy heat plot similar to that shown in Figure 4A , but with mRNAs and CUTs selected to be of similar lengths, 350–700 nt. ( B ) and ( C ) PAR-CLIP occupancy profiles for elongation factors as in Figure 4B and C , with sense mRNAs and divergent antisense CUTs of similar lengths, 350–700 nt, selected from bidirectional promoters. DOI: http://dx.doi.org/10.7554/eLife.25637.010
    Figure Legend Snippet: Asymmetric distribution of EFs at coding and non-coding transcripts of similar length. ( A ) PAR-CLIP occupancy heat plot similar to that shown in Figure 4A , but with mRNAs and CUTs selected to be of similar lengths, 350–700 nt. ( B ) and ( C ) PAR-CLIP occupancy profiles for elongation factors as in Figure 4B and C , with sense mRNAs and divergent antisense CUTs of similar lengths, 350–700 nt, selected from bidirectional promoters. DOI: http://dx.doi.org/10.7554/eLife.25637.010

    Techniques Used: Cross-linking Immunoprecipitation

    28) 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

    29) Product Images from "Dis3l2-Mediated Decay Is a Quality Control Pathway for Noncoding RNAs"

    Article Title: Dis3l2-Mediated Decay Is a Quality Control Pathway for Noncoding RNAs

    Journal: Cell reports

    doi: 10.1016/j.celrep.2016.07.025

    Deep sequencing analysis of the 3′-end of Rmrp RNA (A) Schematic protocol for Rmrp cRACE. See the method section for details. (B) The distribution and length of U-tails in input and FLAG-mutant Dis3l2-IP RNAs. Note the 10–12 nucleotide-long peak in the U-tail length in the Dis3l2-bound Rmrp reads. (C) Analysis of Rmrp 3′ end revealed that U-tails are mainly added to transcripts with 2 additional nucleotides at their 3′ compared to annotated gene. (D) In input samples, majority of Rmrp species are terminated 1 nucleotide before the annotated transcript (highlighted in red), while the U-tailed Rmrp reads in IP samples always end 2 nucleotides downstream.
    Figure Legend Snippet: Deep sequencing analysis of the 3′-end of Rmrp RNA (A) Schematic protocol for Rmrp cRACE. See the method section for details. (B) The distribution and length of U-tails in input and FLAG-mutant Dis3l2-IP RNAs. Note the 10–12 nucleotide-long peak in the U-tail length in the Dis3l2-bound Rmrp reads. (C) Analysis of Rmrp 3′ end revealed that U-tails are mainly added to transcripts with 2 additional nucleotides at their 3′ compared to annotated gene. (D) In input samples, majority of Rmrp species are terminated 1 nucleotide before the annotated transcript (highlighted in red), while the U-tailed Rmrp reads in IP samples always end 2 nucleotides downstream.

    Techniques Used: Sequencing, Mutagenesis

    30) Product Images from "GRID-seq reveals the global RNA-chromatin interactome"

    Article Title: GRID-seq reveals the global RNA-chromatin interactome

    Journal: Nature biotechnology

    doi: 10.1038/nbt.3968

    Characterization of GRID-seq libraries a , Summary of sequenced GRID-seq libraries constructed on two human cell lines (MDA-MB-231 and MM.1S), one mESC, and one Drosophila S2 cells. Shown are raw reads, linker-containing reads, and uniquely mapped reads from mated RNA/DNA pairs. b , Nucleotide frequency of DNA (up) and RNA (bottom) reads. Note specific dinucleotide as part of the AluI recognition site at the 3′ end of DNA reads, but the lack of nucleotide bias in any position of RNA reads. c , d , Strand orientation of mapped RNA (c) and DNA (d) reads. Note the same strand orientation of mapped RNA reads as their transcripts, but not DNA. e , f , Reproducibility of GRID-seq libraries constructed on human (e) and Drosophila (f) cells. g , h , Comparison of GRID-seq detected RNA reads with gene expression detected by RNA-seq of rRNA-depleted total RNA (g) or GRO-seq (h) in Drosophila S2 cells. The lncRNA roX2 is highlighted in both plots. RPK: GRID-seq reads per Kb. RPKM: reads per Kb per million mapped reads.
    Figure Legend Snippet: Characterization of GRID-seq libraries a , Summary of sequenced GRID-seq libraries constructed on two human cell lines (MDA-MB-231 and MM.1S), one mESC, and one Drosophila S2 cells. Shown are raw reads, linker-containing reads, and uniquely mapped reads from mated RNA/DNA pairs. b , Nucleotide frequency of DNA (up) and RNA (bottom) reads. Note specific dinucleotide as part of the AluI recognition site at the 3′ end of DNA reads, but the lack of nucleotide bias in any position of RNA reads. c , d , Strand orientation of mapped RNA (c) and DNA (d) reads. Note the same strand orientation of mapped RNA reads as their transcripts, but not DNA. e , f , Reproducibility of GRID-seq libraries constructed on human (e) and Drosophila (f) cells. g , h , Comparison of GRID-seq detected RNA reads with gene expression detected by RNA-seq of rRNA-depleted total RNA (g) or GRO-seq (h) in Drosophila S2 cells. The lncRNA roX2 is highlighted in both plots. RPK: GRID-seq reads per Kb. RPKM: reads per Kb per million mapped reads.

    Techniques Used: Construct, Multiple Displacement Amplification, Expressing, RNA Sequencing Assay

    31) Product Images from "Adenylylation of small RNA sequencing adapters using the TS2126 RNA ligase I"

    Article Title: Adenylylation of small RNA sequencing adapters using the TS2126 RNA ligase I

    Journal: RNA

    doi: 10.1261/rna.054999.115

    DNA adapters adenylylated using TS2126 Rnl1 are ligated to small RNA acceptors with high efficiency. ( A ) Ligation between adapter 2 adenylylated with TS2126 Rnl1 and the chimeric 5′-DNA/RNA-3′ acceptor 1 using the T4Rnl2trK227Q ligase. The expected product size is 35 nt. ( B ) Ligation of adapters 1T and 2, after adenylylation with TS2126 Rnl1, to RNA acceptor 2. ( C ) DNA adapters adenylylated with TS2126 Rnl1 do not require gel purification prior to ligation to an RNA acceptor. Ligation efficiency comparison of adapter 1T adenylylated using TS2126 Rnl1 with (lane 3 ) or without (lane 2 ) gel purification prior to T4Rnl2trK227Q ligation to acceptor 2. RNA acceptor to DNA adapter molar ratio was 1:2 in all ligations. M, ss DNA size markers; T4Rnl2trK227Q, T4 RNA ligase 2, truncated K227Q mutant from NEB.
    Figure Legend Snippet: DNA adapters adenylylated using TS2126 Rnl1 are ligated to small RNA acceptors with high efficiency. ( A ) Ligation between adapter 2 adenylylated with TS2126 Rnl1 and the chimeric 5′-DNA/RNA-3′ acceptor 1 using the T4Rnl2trK227Q ligase. The expected product size is 35 nt. ( B ) Ligation of adapters 1T and 2, after adenylylation with TS2126 Rnl1, to RNA acceptor 2. ( C ) DNA adapters adenylylated with TS2126 Rnl1 do not require gel purification prior to ligation to an RNA acceptor. Ligation efficiency comparison of adapter 1T adenylylated using TS2126 Rnl1 with (lane 3 ) or without (lane 2 ) gel purification prior to T4Rnl2trK227Q ligation to acceptor 2. RNA acceptor to DNA adapter molar ratio was 1:2 in all ligations. M, ss DNA size markers; T4Rnl2trK227Q, T4 RNA ligase 2, truncated K227Q mutant from NEB.

    Techniques Used: Ligation, Gel Purification, Mutagenesis

    32) Product Images from "Elimination of PCR duplicates in RNA-seq and small RNA-seq using unique molecular identifiers"

    Article Title: Elimination of PCR duplicates in RNA-seq and small RNA-seq using unique molecular identifiers

    Journal: BMC Genomics

    doi: 10.1186/s12864-018-4933-1

    UMI incorporation into small RNA-seq. a Overall workflow. The method uses a 3′ adapter composed of DNA, except for a single, 5′ ribonucleotide (rA); the 5′ adapter is entirely RNA. A standard index barcode allows multiplexing. b Schematic of a read produced from small RNA-seq with UMIs
    Figure Legend Snippet: UMI incorporation into small RNA-seq. a Overall workflow. The method uses a 3′ adapter composed of DNA, except for a single, 5′ ribonucleotide (rA); the 5′ adapter is entirely RNA. A standard index barcode allows multiplexing. b Schematic of a read produced from small RNA-seq with UMIs

    Techniques Used: RNA Sequencing Assay, Multiplexing, Produced

    33) Product Images from "Har-P, a short P-element variant, weaponizes P-transposase to severely impair Drosophila development"

    Article Title: Har-P, a short P-element variant, weaponizes P-transposase to severely impair Drosophila development

    Journal: eLife

    doi: 10.7554/eLife.49948

    Genomic PCR amplifications of P -element insertion loci in HISR-N and HISR-D lines. Characterizing variant distributions among P-element insertions predicted in all HISR-N and two HISR-D genomes ( -D29 and -D43 strains were omitted because > 40 P -element insertions were predicted by TIDAL, Figure 4 ). Loci number represents the predicted P -element insertion sites from TIDAL analysis of Illumina whole genome sequencing. Green arrowheads mark ~0.6 kb Har-P variants, blue arrowheads mark the likely KP -like variant, red arrowheads mark full-length P -elements, and black arrowheads mark full length or uncharacterized P -variants. Quantitation of variants proportions shown in Figure 5D . The similar patterns of amplicons between –D46 and -D51 strains is expected since a majority of the new P -element insertions are shared between these two strains ( Figure 4C ).
    Figure Legend Snippet: Genomic PCR amplifications of P -element insertion loci in HISR-N and HISR-D lines. Characterizing variant distributions among P-element insertions predicted in all HISR-N and two HISR-D genomes ( -D29 and -D43 strains were omitted because > 40 P -element insertions were predicted by TIDAL, Figure 4 ). Loci number represents the predicted P -element insertion sites from TIDAL analysis of Illumina whole genome sequencing. Green arrowheads mark ~0.6 kb Har-P variants, blue arrowheads mark the likely KP -like variant, red arrowheads mark full-length P -elements, and black arrowheads mark full length or uncharacterized P -variants. Quantitation of variants proportions shown in Figure 5D . The similar patterns of amplicons between –D46 and -D51 strains is expected since a majority of the new P -element insertions are shared between these two strains ( Figure 4C ).

    Techniques Used: Polymerase Chain Reaction, Variant Assay, Sequencing, Quantitation Assay

    34) Product Images from "Small RNA Library Preparation Method for Next-Generation Sequencing Using Chemical Modifications to Prevent Adapter Dimer Formation"

    Article Title: Small RNA Library Preparation Method for Next-Generation Sequencing Using Chemical Modifications to Prevent Adapter Dimer Formation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0167009

    Optimization of the 3´ adapter ligation step. Synthetic Let-7d-5p (NNN) miRNA was ligated to the 3´ adapter using the same ligation conditions as the CleanTag library prep workflow step 1. A) Yield increase with addition of PEG 8000 using T4 RNA Ligase 2, truncated KQ and modified 3´ adapter (MP (n-1)). B) Specificity comparison between ligases used in 3´ ligation step: 1) T4 RNA Ligase 2, truncated; 2) T4 RNA Ligase 2, truncated KQ; 3) T4 RNA Ligase 1; 4) No Ligase. Both unmodified and modified (MP (n-1)) 3´ adapters were tested. Side products indicated with red arrows.
    Figure Legend Snippet: Optimization of the 3´ adapter ligation step. Synthetic Let-7d-5p (NNN) miRNA was ligated to the 3´ adapter using the same ligation conditions as the CleanTag library prep workflow step 1. A) Yield increase with addition of PEG 8000 using T4 RNA Ligase 2, truncated KQ and modified 3´ adapter (MP (n-1)). B) Specificity comparison between ligases used in 3´ ligation step: 1) T4 RNA Ligase 2, truncated; 2) T4 RNA Ligase 2, truncated KQ; 3) T4 RNA Ligase 1; 4) No Ligase. Both unmodified and modified (MP (n-1)) 3´ adapters were tested. Side products indicated with red arrows.

    Techniques Used: Ligation, Modification

    35) Product Images from "Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis"

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky067

    Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).
    Figure Legend Snippet: Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).

    Techniques Used: Plasmid Preparation, Amplification, Polymerase Chain Reaction, Clone Assay

    Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.
    Figure Legend Snippet: Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.

    Techniques Used: Sequencing, Polymerase Chain Reaction

    Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).
    Figure Legend Snippet: Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).

    Techniques Used: Plasmid Preparation, Isolation, Purification, Polymerase Chain Reaction, Clone Assay

    36) Product Images from "Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis"

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky067

    Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).
    Figure Legend Snippet: Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).

    Techniques Used: Plasmid Preparation, Amplification, Polymerase Chain Reaction, Clone Assay

    Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.
    Figure Legend Snippet: Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.

    Techniques Used: Sequencing, Polymerase Chain Reaction

    Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).
    Figure Legend Snippet: Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).

    Techniques Used: Plasmid Preparation, Isolation, Purification, Polymerase Chain Reaction, Clone Assay

    37) Product Images from "Addition of non-genomically encoded nucleotides to the 3?-terminus of maize mitochondrial mRNAs: truncated rps12 mRNAs frequently terminate with CCA"

    Article Title: Addition of non-genomically encoded nucleotides to the 3?-terminus of maize mitochondrial mRNAs: truncated rps12 mRNAs frequently terminate with CCA

    Journal: Nucleic Acids Research

    doi:

    Amplification of anchor-ligated cDNAs is dependent on T4 RNA ligase. Maize mitochondrial RNA (1–2 µg) was incubated with 40 pmol of anchor oligonucleotide in the presence or absence of T4 RNA ligase. Anchor-ligated RNAs were reverse transcribed and amplified by PCR and the cDNA products were electrophoresed on agarose gels. Lanes marked M show the migration of commercial DNA size markers. PCR products for the following cDNAs are shown: ( A ) atp9 , lanes 1 and 2; ( B ) cox2 , lanes 3 and 4; ( C ) rps12 , lanes 5 and 6, and trnS , lanes 7 and 8. Amplification of anchor-ligated atp9 . Odd numbered lanes (1, 3, 5 and 7) included T4 RNA ligase and even numbered lanes (2, 4, 6 and 8) omitted T4 RNA ligase.
    Figure Legend Snippet: Amplification of anchor-ligated cDNAs is dependent on T4 RNA ligase. Maize mitochondrial RNA (1–2 µg) was incubated with 40 pmol of anchor oligonucleotide in the presence or absence of T4 RNA ligase. Anchor-ligated RNAs were reverse transcribed and amplified by PCR and the cDNA products were electrophoresed on agarose gels. Lanes marked M show the migration of commercial DNA size markers. PCR products for the following cDNAs are shown: ( A ) atp9 , lanes 1 and 2; ( B ) cox2 , lanes 3 and 4; ( C ) rps12 , lanes 5 and 6, and trnS , lanes 7 and 8. Amplification of anchor-ligated atp9 . Odd numbered lanes (1, 3, 5 and 7) included T4 RNA ligase and even numbered lanes (2, 4, 6 and 8) omitted T4 RNA ligase.

    Techniques Used: Amplification, Incubation, Polymerase Chain Reaction, Migration

    38) Product Images from "T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis"

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1169

    The schematic of the TEDA method. The blue half-moon represents T5 exonuclease. The double lined rectangle with a gap represents a linearized plasmid. The double vertical lines represent the insert DNA. Lines with same color indicate the homologous region. Step 1: T5 exonuclease cuts from the 5′ ends of linearized plasmid and insert DNA to generate 5′-overhangs. Step 2: the 5′-overhangs anneal to each other. Step 3: The cyclized DNA with DNA gaps is transformed into cells and the gaps are repaired in vivo .
    Figure Legend Snippet: The schematic of the TEDA method. The blue half-moon represents T5 exonuclease. The double lined rectangle with a gap represents a linearized plasmid. The double vertical lines represent the insert DNA. Lines with same color indicate the homologous region. Step 1: T5 exonuclease cuts from the 5′ ends of linearized plasmid and insert DNA to generate 5′-overhangs. Step 2: the 5′-overhangs anneal to each other. Step 3: The cyclized DNA with DNA gaps is transformed into cells and the gaps are repaired in vivo .

    Techniques Used: Plasmid Preparation, Transformation Assay, In Vivo

    Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Clone Assay

    Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Plasmid Preparation, Clone Assay, Transformation Assay

    39) Product Images from "NEAT1 Scaffolds RNA Binding Proteins and the Microprocessor to Globally Enhance Pri-miRNA Processing"

    Article Title: NEAT1 Scaffolds RNA Binding Proteins and the Microprocessor to Globally Enhance Pri-miRNA Processing

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.3455

    Localization of induced pri-miR-1 in paraspeckles in differentiated C2C12 cells and the proposed bird nest model ( a ) The expression levels of NEAT1 and pri-miR-1 quantified by RT-qPCR in undifferentiated (unDF) and differentiated (DF) C2C12 cells. Inset shows the induction of the differentiation marker MHC and MYOG by Western blotting. ( b ) Enhanced paraspeckles after C2C12 differentiation, detected by NONO immunostaining. ( c ) FISH analysis of inducible pri-miR-1. No pri-miR-1 signal was detectable in C2C12 cells before differentiation and colocalization of induced pri-miR-1 with NONO on paraspeckles in differentiated C2C12 cells. ( d ) Colocalization of induced pri-miR-1 with DGCR8 in differentiated C2C12 cells. Scale bars in b , c , d : 10 μm. ( e ) The proposed bird nest model for NEAT1- orchestrated enhancement of pri-miRNA processing by the Microprocessor. Multiple RBPs, including the NONO-PSF heterodimer, extensively interact with NEAT1 _ V2 , on which additional NEAT1_V1 and NEAT1_V2 may be added to build a bird nest-like structure. Various RBPs may also bring pri-miRNAs to the nest and various RNA secondary structures in NEAT1 , including a poorly processed pri-miR-612 near the 3′ end of NEAT1_V2 , may help recruit the Microprocessor. These NEAT1 -containing RNPs may exist in both the microscopic form (left) and become “aggregated” to generate larger structures visible as paraspeckles (right). In both forms, such RNA-orchestrated structures may create the proximity between pri-miRNAs and the Microprocessor to enhance the kinetics of pri-miRNA processing. Bar graphs in a are presented as mean ± SEM (n=3, technical replicates). ***P
    Figure Legend Snippet: Localization of induced pri-miR-1 in paraspeckles in differentiated C2C12 cells and the proposed bird nest model ( a ) The expression levels of NEAT1 and pri-miR-1 quantified by RT-qPCR in undifferentiated (unDF) and differentiated (DF) C2C12 cells. Inset shows the induction of the differentiation marker MHC and MYOG by Western blotting. ( b ) Enhanced paraspeckles after C2C12 differentiation, detected by NONO immunostaining. ( c ) FISH analysis of inducible pri-miR-1. No pri-miR-1 signal was detectable in C2C12 cells before differentiation and colocalization of induced pri-miR-1 with NONO on paraspeckles in differentiated C2C12 cells. ( d ) Colocalization of induced pri-miR-1 with DGCR8 in differentiated C2C12 cells. Scale bars in b , c , d : 10 μm. ( e ) The proposed bird nest model for NEAT1- orchestrated enhancement of pri-miRNA processing by the Microprocessor. Multiple RBPs, including the NONO-PSF heterodimer, extensively interact with NEAT1 _ V2 , on which additional NEAT1_V1 and NEAT1_V2 may be added to build a bird nest-like structure. Various RBPs may also bring pri-miRNAs to the nest and various RNA secondary structures in NEAT1 , including a poorly processed pri-miR-612 near the 3′ end of NEAT1_V2 , may help recruit the Microprocessor. These NEAT1 -containing RNPs may exist in both the microscopic form (left) and become “aggregated” to generate larger structures visible as paraspeckles (right). In both forms, such RNA-orchestrated structures may create the proximity between pri-miRNAs and the Microprocessor to enhance the kinetics of pri-miRNA processing. Bar graphs in a are presented as mean ± SEM (n=3, technical replicates). ***P

    Techniques Used: Expressing, Quantitative RT-PCR, Marker, Western Blot, Immunostaining, Fluorescence In Situ Hybridization

    Involvement of paraspeckle-associated proteins and lncRNA in pri-miRNA processing ( a ) Coomassie brilliant blue staining of proteins captured by individual pri-miR-17–92a from HeLa nuclear extracts. Specific proteins identified by mass spectrometry are indicated on the right. ( b ) Knockdown of three paraspeckle-associated proteins, NEAT1_V2 and MALAT1 , respectively, quantified by Western blotting and RT-qPCR. ( c ) The expression of pri-miR-17–92a (left) and individual mature miRNAs from the pri-miR-17–92a locus (right) in response to knockdown of paraspeckle-associated factors and NEAT1 , determined by RT-qPCR. ( d ) RT-qPCR was performed to confirm NEAT1 knockout (KO) with CRISPR/Cas and their impact on miRNA expression. ( e ) miRNA profiling in response to specific knockdowns as in c relative to control treated with siRNA against GFP. Color key on top indicates changes in log2 scale. ( f ) Summary of up-regulated (≥1.5-fold), no change, or down-regulated (≥1.5-fold) numbers of miRNAs based on small RNA-seq in response to specific knockdowns as in e . Uncropped images of Western blots in b are shown in Supplementary Data Set 1 . Data in b , c,d are presented as mean ± SEM (n=3, technical replicates). *P
    Figure Legend Snippet: Involvement of paraspeckle-associated proteins and lncRNA in pri-miRNA processing ( a ) Coomassie brilliant blue staining of proteins captured by individual pri-miR-17–92a from HeLa nuclear extracts. Specific proteins identified by mass spectrometry are indicated on the right. ( b ) Knockdown of three paraspeckle-associated proteins, NEAT1_V2 and MALAT1 , respectively, quantified by Western blotting and RT-qPCR. ( c ) The expression of pri-miR-17–92a (left) and individual mature miRNAs from the pri-miR-17–92a locus (right) in response to knockdown of paraspeckle-associated factors and NEAT1 , determined by RT-qPCR. ( d ) RT-qPCR was performed to confirm NEAT1 knockout (KO) with CRISPR/Cas and their impact on miRNA expression. ( e ) miRNA profiling in response to specific knockdowns as in c relative to control treated with siRNA against GFP. Color key on top indicates changes in log2 scale. ( f ) Summary of up-regulated (≥1.5-fold), no change, or down-regulated (≥1.5-fold) numbers of miRNAs based on small RNA-seq in response to specific knockdowns as in e . Uncropped images of Western blots in b are shown in Supplementary Data Set 1 . Data in b , c,d are presented as mean ± SEM (n=3, technical replicates). *P

    Techniques Used: Staining, Mass Spectrometry, Western Blot, Quantitative RT-PCR, Expressing, Knock-Out, CRISPR, RNA Sequencing Assay

    Genome-wide analysis of NONO-PSF-RNA interactions ( a ) Immunoprecipitated NONO-PSF crosslinked to RNA. The complex was treated with two different concentrations of MNase (1:1,000 or 1:50,000 dilution); RNA in the complex was 32 p-labeled with T4 polynucleotide kinase. Proteins and RNA were visualized by Western blotting (left) and autoradiography (right). Indicated bands (right) were individually isolated for CLIP-seq library construction. ( b ) Venn Diagram showing overlapped pri-miRNAs bound by NONO and PSF in HeLa cells. ( c ) Footprint of NONO and PSF on pri-miRNAs. ( d ) Representative NONO and PSF binding tracks on the pri-miR-17–92a transcript. ( e ) The binding profiles of NONO and PSF on NEAT1 in comparison with the published DGCR8 CLIP-seq signals 44 . Y-axis in d and e shows CLIP-seq read density in each case. The region encoding for miR-612 is indicated at bottom. Uncropped images of Western blots and autoradiography in a are shown in Supplementary Data Set 1 .
    Figure Legend Snippet: Genome-wide analysis of NONO-PSF-RNA interactions ( a ) Immunoprecipitated NONO-PSF crosslinked to RNA. The complex was treated with two different concentrations of MNase (1:1,000 or 1:50,000 dilution); RNA in the complex was 32 p-labeled with T4 polynucleotide kinase. Proteins and RNA were visualized by Western blotting (left) and autoradiography (right). Indicated bands (right) were individually isolated for CLIP-seq library construction. ( b ) Venn Diagram showing overlapped pri-miRNAs bound by NONO and PSF in HeLa cells. ( c ) Footprint of NONO and PSF on pri-miRNAs. ( d ) Representative NONO and PSF binding tracks on the pri-miR-17–92a transcript. ( e ) The binding profiles of NONO and PSF on NEAT1 in comparison with the published DGCR8 CLIP-seq signals 44 . Y-axis in d and e shows CLIP-seq read density in each case. The region encoding for miR-612 is indicated at bottom. Uncropped images of Western blots and autoradiography in a are shown in Supplementary Data Set 1 .

    Techniques Used: Genome Wide, Immunoprecipitation, Labeling, Western Blot, Autoradiography, Isolation, Cross-linking Immunoprecipitation, Binding Assay

    NEAT1 -mediated interaction networks for enhancing pri-miRNA processing ( a ) Illustration of NEAT1_V1, NEAT1_V2 and derived RNA fragments. Highlighted on the right are pri-miR-612 near the 3′ end of NEAT1_V2 and the 3′ fragments before (3′F) or after deletion of the pre-miR-612 stem-loop (3′F-DS). ( b , c ) Enhanced processing of the pri-miR-17–92a reporter by NEAT_V1 and 3′F, but not a middle fragment from NEAT1_V2 (midF) or 3′F-DS (left panels). The right panels show RNA pulldown results from HeLa nuclear extracts, analyzed by Western blotting for NONO-PSF and DGCR8. ( d ) Knockdown of NEAT1_V2 diminished the enhancement of pri-miRNA processing by overexpressed NEAT_V1 on the pri-miR-17–92a (left) or pri-Let-7b (right) processing reporter. ( e ) Knockout of NEAT1_V2 prevented the enhancement of pri-miRNA processing by overexpressed NEAT_V1 and the 3′F on the pri-miR-17–92a processing reporter. Bar graphs in b , c , d , and e are presented as mean ± SEM (n=3, cell culture). *P
    Figure Legend Snippet: NEAT1 -mediated interaction networks for enhancing pri-miRNA processing ( a ) Illustration of NEAT1_V1, NEAT1_V2 and derived RNA fragments. Highlighted on the right are pri-miR-612 near the 3′ end of NEAT1_V2 and the 3′ fragments before (3′F) or after deletion of the pre-miR-612 stem-loop (3′F-DS). ( b , c ) Enhanced processing of the pri-miR-17–92a reporter by NEAT_V1 and 3′F, but not a middle fragment from NEAT1_V2 (midF) or 3′F-DS (left panels). The right panels show RNA pulldown results from HeLa nuclear extracts, analyzed by Western blotting for NONO-PSF and DGCR8. ( d ) Knockdown of NEAT1_V2 diminished the enhancement of pri-miRNA processing by overexpressed NEAT_V1 on the pri-miR-17–92a (left) or pri-Let-7b (right) processing reporter. ( e ) Knockout of NEAT1_V2 prevented the enhancement of pri-miRNA processing by overexpressed NEAT_V1 and the 3′F on the pri-miR-17–92a processing reporter. Bar graphs in b , c , d , and e are presented as mean ± SEM (n=3, cell culture). *P

    Techniques Used: Derivative Assay, Western Blot, Knock-Out, Cell Culture

    40) Product Images from "Elimination of PCR duplicates in RNA-seq and small RNA-seq using unique molecular identifiers"

    Article Title: Elimination of PCR duplicates in RNA-seq and small RNA-seq using unique molecular identifiers

    Journal: BMC Genomics

    doi: 10.1186/s12864-018-4933-1

    UMI incorporation into small RNA-seq. a Overall workflow. The method uses a 3′ adapter composed of DNA, except for a single, 5′ ribonucleotide (rA); the 5′ adapter is entirely RNA. A standard index barcode allows multiplexing. b Schematic of a read produced from small RNA-seq with UMIs
    Figure Legend Snippet: UMI incorporation into small RNA-seq. a Overall workflow. The method uses a 3′ adapter composed of DNA, except for a single, 5′ ribonucleotide (rA); the 5′ adapter is entirely RNA. A standard index barcode allows multiplexing. b Schematic of a read produced from small RNA-seq with UMIs

    Techniques Used: RNA Sequencing Assay, Multiplexing, Produced

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    Amplification:

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    Article Snippet: .. Other DNA fragments were obtained by PCR with the Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific, Waltham, MA USA), except for the fragment containing the stem-loop (S-SL), which was obtained with the LA Taq DNA polymerase (Takara Bio Europe) using the GC buffer I. PCR products were purified using either the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany), the Wizard® SV Gel and PCR Clean-Up System (Promega GmbH, Mannheim, Germany) or the Monarch™ PCR & DNA Cleanup Kit (NEB). .. Unless otherwise stated, plasmids were obtained from the GeneArt Gene Synthesis Service (Thermo Fisher Scientific).

    Article Title: A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters
    Article Snippet: .. The amplicons were purified with NEB Monarch PCR & DNA Cleanup Kit and quantified with Nanodrop. .. 200 ng were checked on a gel and 500 ng were sent to GENEWIZ to be sequenced with NGS-based amplicon sequencing.

    Article Title: Single telomere length analysis in Ustilago maydis, a high-resolution tool for examining fungal telomere length distribution and C-strand 5’-end processing
    Article Snippet: .. Following PCR amplification, the DNA was isolated using the Monarch® PCR & DNA Cleanup Kit (NEB, Inc.) and then introduced into the pMiniT 2.0 vector using the NEB® PCR Cloning Kit. ..

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis
    Article Snippet: .. After 25 μl pilot PCRs, PCRs were scaled up (usually to 2 × 50 μl reactions) and gel purified using Monarch PCR and DNA cleanup kits (NEB). ..

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis
    Article Snippet: .. PCR products were purified using GeneJET PCR Purification Kits (Thermo Fisher Scientific, Waltham MA, USA), Nucleospin Gel and PCR Clean-up (Machery-Nagel GmbH, Düren, Germany) or Monarch PCR and DNA Cleanup kits (NEB). .. Gel purification was carried out using Monarch DNA Gel Extraction Kit (NEB).

    Gel Extraction:

    Article Title: Selection of an Efficient AAV Vector for Robust CNS Transgene Expression
    Article Snippet: .. The amplicons were then purified (Monarch PCR and DNA cleanup kit, New England Biolabs), digested by KpnI, AgeI, and BanII, and the Cap9 KpnI-AgeI fragments (144 bp) were agarose gel purified (Monarch DNA gel extraction kit, New England Biolabs) before ligation in the pUC57-Cap9-XbaI/AgeI/KpnI plasmid (opened with KpnI and AgeI and dephosphorylated with calf inositol phosphatase, New England Biolabs). .. The ligation products were transformed into electrocompetent DH5α bacteria (New England Biolabs), and the entire transformation was grown overnight in Lysogeny broth (LB)-ampicillin medium. pUC57-Cap9-XbaI/AgeI/KpnI plasmid was purified by Maxi Prep (QIAGEN).

    Chromatin Immunoprecipitation:

    Article Title: Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application
    Article Snippet: .. The ChIP DNA Clean & Concentrator™ (Zymo Research; Zy), the Monarch® PCR & DNA Cleanup Kit (New England Biolabs; Ne), the MinElute PCR Purification Kit (Qiagen, Qm), the QIAquick PCR Purification Kit (Qiagen; Qp), the Agencourt AMPure XP kit (Beckman; Ba) and the RNAClean™ XP kit (Beckman; Br), and phenol/chloroform extraction (Invitrogen; PC) performed well with de-crosslinked chromatin. .. These reagents recovered 78.1% to 95.7% with 10–50 ng of purified DNA, 81.7% to 96.8% with 5 ng of DNA, and 68.1% to 82.9% with 1 ng of DNA except phenol/chloroform extraction with over 100%.

    Plasmid Preparation:

    Article Title: Selection of an Efficient AAV Vector for Robust CNS Transgene Expression
    Article Snippet: .. The amplicons were then purified (Monarch PCR and DNA cleanup kit, New England Biolabs), digested by KpnI, AgeI, and BanII, and the Cap9 KpnI-AgeI fragments (144 bp) were agarose gel purified (Monarch DNA gel extraction kit, New England Biolabs) before ligation in the pUC57-Cap9-XbaI/AgeI/KpnI plasmid (opened with KpnI and AgeI and dephosphorylated with calf inositol phosphatase, New England Biolabs). .. The ligation products were transformed into electrocompetent DH5α bacteria (New England Biolabs), and the entire transformation was grown overnight in Lysogeny broth (LB)-ampicillin medium. pUC57-Cap9-XbaI/AgeI/KpnI plasmid was purified by Maxi Prep (QIAGEN).

    Article Title: Single telomere length analysis in Ustilago maydis, a high-resolution tool for examining fungal telomere length distribution and C-strand 5’-end processing
    Article Snippet: .. Following PCR amplification, the DNA was isolated using the Monarch® PCR & DNA Cleanup Kit (NEB, Inc.) and then introduced into the pMiniT 2.0 vector using the NEB® PCR Cloning Kit. ..

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    New England Biolabs t4 rna ligase
    Amplification of anchor-ligated cDNAs is dependent on <t>T4</t> RNA ligase. Maize mitochondrial RNA (1–2 µg) was incubated with 40 pmol of anchor oligonucleotide in the presence or absence of T4 RNA ligase. Anchor-ligated RNAs were reverse transcribed and amplified by PCR and the cDNA products were electrophoresed on agarose gels. Lanes marked M show the migration of commercial DNA size markers. PCR products for the following cDNAs are shown: ( A ) atp9 , lanes 1 and 2; ( B ) cox2 , lanes 3 and 4; ( C ) rps12 , lanes 5 and 6, and trnS , lanes 7 and 8. Amplification of anchor-ligated atp9 . Odd numbered lanes (1, 3, 5 and 7) included T4 RNA ligase and even numbered lanes (2, 4, 6 and 8) omitted T4 RNA ligase.
    T4 Rna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs t5 exonuclease
    The schematic of the TEDA method. The blue half-moon represents T5 exonuclease. The double lined rectangle with a gap represents a linearized plasmid. The double vertical lines represent the insert DNA. Lines with same color indicate the homologous region. Step 1: <t>T5</t> exonuclease cuts from the 5′ ends of linearized plasmid and insert DNA to generate 5′-overhangs. Step 2: the 5′-overhangs anneal to each other. Step 3: The cyclized DNA with DNA gaps is transformed into cells and the gaps are repaired in vivo .
    T5 Exonuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs peg 8000
    The schematic of the TEDA method. The blue half-moon represents T5 exonuclease. The double lined rectangle with a gap represents a linearized plasmid. The double vertical lines represent the insert DNA. Lines with same color indicate the homologous region. Step 1: <t>T5</t> exonuclease cuts from the 5′ ends of linearized plasmid and insert DNA to generate 5′-overhangs. Step 2: the 5′-overhangs anneal to each other. Step 3: The cyclized DNA with DNA gaps is transformed into cells and the gaps are repaired in vivo .
    Peg 8000, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 91/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Amplification of anchor-ligated cDNAs is dependent on T4 RNA ligase. Maize mitochondrial RNA (1–2 µg) was incubated with 40 pmol of anchor oligonucleotide in the presence or absence of T4 RNA ligase. Anchor-ligated RNAs were reverse transcribed and amplified by PCR and the cDNA products were electrophoresed on agarose gels. Lanes marked M show the migration of commercial DNA size markers. PCR products for the following cDNAs are shown: ( A ) atp9 , lanes 1 and 2; ( B ) cox2 , lanes 3 and 4; ( C ) rps12 , lanes 5 and 6, and trnS , lanes 7 and 8. Amplification of anchor-ligated atp9 . Odd numbered lanes (1, 3, 5 and 7) included T4 RNA ligase and even numbered lanes (2, 4, 6 and 8) omitted T4 RNA ligase.

    Journal: Nucleic Acids Research

    Article Title: Addition of non-genomically encoded nucleotides to the 3?-terminus of maize mitochondrial mRNAs: truncated rps12 mRNAs frequently terminate with CCA

    doi:

    Figure Lengend Snippet: Amplification of anchor-ligated cDNAs is dependent on T4 RNA ligase. Maize mitochondrial RNA (1–2 µg) was incubated with 40 pmol of anchor oligonucleotide in the presence or absence of T4 RNA ligase. Anchor-ligated RNAs were reverse transcribed and amplified by PCR and the cDNA products were electrophoresed on agarose gels. Lanes marked M show the migration of commercial DNA size markers. PCR products for the following cDNAs are shown: ( A ) atp9 , lanes 1 and 2; ( B ) cox2 , lanes 3 and 4; ( C ) rps12 , lanes 5 and 6, and trnS , lanes 7 and 8. Amplification of anchor-ligated atp9 . Odd numbered lanes (1, 3, 5 and 7) included T4 RNA ligase and even numbered lanes (2, 4, 6 and 8) omitted T4 RNA ligase.

    Article Snippet: The ligation reactions contained 50 mM Tris–HCl, pH 8.0, 10 mM MgCl2 , 0.2 mg/ml BSA, 1 mM hexamine cobalt chloride, 20 µM ATP, 12.5% PEG 8000 and 15 U T4 RNA ligase (New England Biolabs).

    Techniques: Amplification, Incubation, Polymerase Chain Reaction, Migration

    The schematic of the TEDA method. The blue half-moon represents T5 exonuclease. The double lined rectangle with a gap represents a linearized plasmid. The double vertical lines represent the insert DNA. Lines with same color indicate the homologous region. Step 1: T5 exonuclease cuts from the 5′ ends of linearized plasmid and insert DNA to generate 5′-overhangs. Step 2: the 5′-overhangs anneal to each other. Step 3: The cyclized DNA with DNA gaps is transformed into cells and the gaps are repaired in vivo .

    Journal: Nucleic Acids Research

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis

    doi: 10.1093/nar/gky1169

    Figure Lengend Snippet: The schematic of the TEDA method. The blue half-moon represents T5 exonuclease. The double lined rectangle with a gap represents a linearized plasmid. The double vertical lines represent the insert DNA. Lines with same color indicate the homologous region. Step 1: T5 exonuclease cuts from the 5′ ends of linearized plasmid and insert DNA to generate 5′-overhangs. Step 2: the 5′-overhangs anneal to each other. Step 3: The cyclized DNA with DNA gaps is transformed into cells and the gaps are repaired in vivo .

    Article Snippet: For optimization, PEG 8000 and the proper dilution of T5 exonuclease were two key factors for TEDA (Figure ).

    Techniques: Plasmid Preparation, Transformation Assay, In Vivo

    Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.

    Journal: Nucleic Acids Research

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis

    doi: 10.1093/nar/gky1169

    Figure Lengend Snippet: Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.

    Article Snippet: For optimization, PEG 8000 and the proper dilution of T5 exonuclease were two key factors for TEDA (Figure ).

    Techniques: Clone Assay

    Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.

    Journal: Nucleic Acids Research

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis

    doi: 10.1093/nar/gky1169

    Figure Lengend Snippet: Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.

    Article Snippet: For optimization, PEG 8000 and the proper dilution of T5 exonuclease were two key factors for TEDA (Figure ).

    Techniques: Plasmid Preparation, Clone Assay, Transformation Assay