peg 8000  (New England Biolabs)


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  • 99
    Name:
    T4 RNA Ligase 2 truncated K227Q
    Description:
    T4 RNA Ligase 2 truncated K227Q 10 000 units
    Catalog Number:
    M0351L
    Price:
    273
    Size:
    10 000 units
    Category:
    RNA Ligases
    Score:
    85
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    Structured Review

    New England Biolabs peg 8000
    T4 RNA Ligase 2 truncated K227Q
    T4 RNA Ligase 2 truncated K227Q 10 000 units
    https://www.bioz.com/result/peg 8000/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    peg 8000 - by Bioz Stars, 2020-01
    99/100 stars

    Images

    1) 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

    Related Articles

    Clone Assay:

    Article Title: Preparation of Multiplexed Small RNA Libraries From Plants
    Article Snippet: Fiber glass (Corning Incorporated, catalog number: 988-10144) Extra thick blot paper (Bio-Rad Laboratories, catalog number: 170-3969) Whatman DE81 ion exchange cellulose chromatography paper (Thermo Fisher Scientific, catalog number: 05-717-1A) GlycoBlue coprecipitant (Life Technologies, catalog number: AM9516) 100% ethanol 75% ethanol EB buffer (QIAGEN, catalog number: 19086) Qubit RNA HS Assay Kit for quantification of purified small RNAs (Life Technologies, catalog number: ) Qubit dsDNA HS Assay Kit for quantification of DNA amplicon (Life Technologies, catalog number: ) Low salt buffer (see Recipes) High salt buffer (see Recipes) .. 8-Strip PCR thin-walled, 200 μl tubes (Corning Incorporated, Axygen® , catalog number: PCR-0208-CP-C) miRNA cloning linker 1 (/5′App/CTGTAGGCACCATCAAT/3′ddC/; 1 nm) (Integrated DNA Technologies, catalog number: 11-04-03-05) Truncated, K227Q mutation T4 RNA Ligase 2 (New England Biolabs, catalog number: M0351L) De-adenylase (New England Biolabs, catalog number: M0331S) Exonuclease VII (United State Biological, catalog number: 70082Z) RNA 5′ Adapter (GUUCAGAGUUCUACAGUCCGACGAUC) (Illumina RA5, catalog number: 15013205) dATP (Life Technologies, catalog number: 55082) T4 RNA Ligase I (Life Technologies, catalog number: AM2141) RT-PCR Primer (ATTGATGGTGCCTACAG; 25 nmol; de-salted) (Integrated DNA Technologies) SuperScript III (Life Technologies, catalog number: 18080051) Phusion High Fidelity II (Thermo Fisher Scientific, catalog number: F549L) 5′ PCR Primer (Illumina small RNA PCR primer 2: AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA) 3′ Indexed PCR Primer I1 – I12 (100 nmol; PAGE-purified) (Integrated DNA Technologies) Note: The barcodes below (underlined sequences) are reverse complemented in the final Illumina output sequence (see sequence in square brackets for each primer). .. I1 [CGATGT]: CAAGCAGAAGACGGCATACGA ACATCG ATTGATGGTGCCTACAG I2 [GATCAC]: CAAGCAGAAGACGGCATACGA GTGATC ATTGATGGTGCCTACAG I3 [CAGATG]: CAAGCAGAAGACGGCATACGA CATCTG ATTGATGGTGCCTACAG I4 [TACGTT]: CAAGCAGAAGACGGCATACGA AACGTA ATTGATGGTGCCTACAG I5 [TTACCA]: CAAGCAGAAGACGGCATACGA TGGTAA ATTGATGGTGCCTACAG I6 [ACTGTA]: CAAGCAGAAGACGGCATACGA TACAGT ATTGATGGTGCCTACAG I7 [ATCACG]: CAAGCAGAAGACGGCATACGA CGTGAT ATTGATGGTGCCTACAG I8 [ACTTGT]: CAAGCAGAAGACGGCATACGA ACAAGT ATTGATGGTGCCTACAG I9 [GCCAAT]: CAAGCAGAAGACGGCATACGA ATTGGC ATTGATGGTGCCTACAG I10 [TGCTAG]: CAAGCAGAAGACGGCATACGA CTAGCA ATTGATGGTGCCTACAG I11 [CTTGTA]: CAAGCAGAAGACGGCATACGA TACAAG ATTGATGGTGCCTACAG I12 [TCAGGC]: CAAGCAGAAGACGGCATACGA GCCTGA ATTGATGGTGCCTACAG QIAquick PCR Purification Kit (QIAGEN, catalog number: 28106) Diethylpyrocarbonate (DEPC)-H2 O/ RNase-free H2 O DNA size marker suitable for detecting a ~120 bp fragment (50 bp step ladder, Promega Corporation, catalog number: G4521; 25 bp step ladder, Promega Corporation, catalog number: G4511) 3 M sodium acetate (NaOAc) (pH 5.5) Reagents for 6% native PAGE 37.5: 1 polyacrylamide:bisacrylamide (see Recipes) TEMED (Life Technologies, catalog number: 15524-010) 10% Ammonium persulfate (Sigma, catalog number: A3678-100G) (See Recipes)

    Article Title: The Coding Regions of Germline mRNAs Confer Sensitivity to Argonaute Regulation in C. elegans
    Article Snippet: Paragraph title: Small RNA Cloning and Deep Sequencing ... Samples from wild-type and mutant were pretreated with a homemade 5′ polyphosphatase and were ligated to the 3′ adaptor linker 1 (5′ rAppAGATCGGAAGAGCACACGTCTGAACTCCAGTCA/3ddC/3′, IDT) using T4 RNA ligase 2 (M0351S, NEB).

    Article Title: Evaluating bias-reducing protocols for RNA sequencing library preparation
    Article Snippet: For the CircLig libraries, 40 ng RNA pool (~6 pmol) and 100 ng Universal Cloning Linker (~18 pmol; NEB) were denatured at 80°C for 2 min then placed immediately on ice. .. Ligation with 200 U T4 RNA Ligase 2 truncated K227Q mutant (trRnl2 K227Q; NEB) was performed in the presence of 1X RNA ligase buffer (NEB), 15% PEG8000, 20 U SuperaseIn (Ambion), at 25°C for 3 hr.

    Article Title: The microRNAs in an Ancient Protist Repress the Variant-Specific Surface Protein Expression by Targeting the Entire Coding Sequence
    Article Snippet: For the addition of a sonication step to the procedure, the cells were resuspend in RIPA buffer as described previously and sonicated twice with five 1 second bursts at a 20% duty cycle. .. The purified small RNAs were ligated at the 3′-end to 5–10 µM gel purified miRNA Cloning Linker-1 (IDT) using T4 RNA ligase 2 and truncated with K227Q (NEB) by incubating at 25°C for 1 hr, 15°C for 2 hrs and followed by a final 4°C incubation overnight. .. The reaction mixture was analyzed in 15% polyacrylamide/8 M Urea gel, and the band of ligated product was excised.

    Article Title: Transgenerational function of Tetrahymena Piwi protein Twi8p at distinctive noncoding RNA loci
    Article Snippet: Size selection of purified sRNAs was performed by denaturing PAGE. sRNAs were prepared for Solexa sequencing as previously described ( ). .. Briefly, IDT miRNA cloning linker 1 (Modban) was ligated to the 3′ end of the purified sRNAs using T4 RNA ligase, truncated, K227Q (NEB). .. Ligations were incubated at 16°C overnight to minimize the impact of 3′-terminal 2′ O -methylation on the sRNAs ( ).

    Centrifugation:

    Article Title: A Novel Epigenetic Silencing Pathway Involving the Highly Conserved 5’-3’ Exoribonuclease Dhp1/Rat1/Xrn2 in Schizosaccharomyces pombe
    Article Snippet: The TEV eluates were collected by centrifugation and incubated with 10U of Turbo DNase (Ambion) for 8 min at 37°C followed by incubation with RNase Cocktail Enzyme Mix (Ambion; 0.005 U RnaseA, 0.2 U Rnase T1) for 2 min at 37°C. .. All washes, alkaline phosphatase treatment and 3’ linker ligation were carried out as described except that 40U T4 RNA ligase 2 truncated K227Q (NEB) was used instead of T4 RNA ligase.

    Amplification:

    Article Title: The Gcn4 transcription factor reduces protein synthesis capacity and extends yeast lifespan
    Article Snippet: Ligation of RNA 3′ adapter (RA3, part # 15013207, Illumina) was done using T4 RNA Ligase 2, truncated K227Q (M0351L, New England Biolabs Inc) according to the Illumina protocol. .. Complementary DNA (cDNA) was synthesized using RNA RT Primer (RTP, part #15013981, Illumina) and SuperScript III (18080044, Invitrogen) as per Illumina protocol.

    Article Title: The Coding Regions of Germline mRNAs Confer Sensitivity to Argonaute Regulation in C. elegans
    Article Snippet: Samples from wild-type and mutant were pretreated with a homemade 5′ polyphosphatase and were ligated to the 3′ adaptor linker 1 (5′ rAppAGATCGGAAGAGCACACGTCTGAACTCCAGTCA/3ddC/3′, IDT) using T4 RNA ligase 2 (M0351S, NEB). .. Samples from wild-type and mutant were pretreated with a homemade 5′ polyphosphatase and were ligated to the 3′ adaptor linker 1 (5′ rAppAGATCGGAAGAGCACACGTCTGAACTCCAGTCA/3ddC/3′, IDT) using T4 RNA ligase 2 (M0351S, NEB).

    Article Title: Substantial and robust changes in microRNA transcriptome support postnatal development of the hypothalamus in rat
    Article Snippet: Individual cDNA libraries were built following an Illumina-like protocol in which 3′- and 5′-adapters were sequentially ligated at the 3′ and 5′ ends of small RNAs, respectively, to allow for reverse transcription (RT) and amplification by polymerase chain reaction (PCR). .. To prevent secondary structures, mix were heated for 3 min at 70 °C, then kept on ice while adding [50%] PEG 8000 (2.4 ul), 80 U of truncated T4 RNA ligase 2 K227Q (0.4 ul) and the corresponding buffer (1 ul) (all provided by NEB).

    Article Title: The ribonuclease activity of SAMHD1 is required for HIV-1 restriction
    Article Snippet: 3′ adaptor (5′/rApp/TGGAATTCTCGGGTGCCAAGG/ddC/-3′, IDT) was ligated using T4 RNA ligase truncated K227Q (NEB) followed by ligation of 5′ adaptor (5′ Solexa linker, 100-M; 5′rGrUrUrCrArGrArGrUrUrCrUrArCrArGrUrCrCrGrArCrGrA rUrC-3′, IDT) using T4 RNA ligase (TaKaRa). .. 3′ adaptor (5′/rApp/TGGAATTCTCGGGTGCCAAGG/ddC/-3′, IDT) was ligated using T4 RNA ligase truncated K227Q (NEB) followed by ligation of 5′ adaptor (5′ Solexa linker, 100-M; 5′rGrUrUrCrArGrArGrUrUrCrUrArCrArGrUrCrCrGrArCrGrA rUrC-3′, IDT) using T4 RNA ligase (TaKaRa).

    Article Title: Single‐cell mRNA profiling reveals the hierarchical response of mi RNA targets to mi RNA induction
    Article Snippet: After another round of RNeasy MinElute Cleanup Kit (Qiagen, Inc.), a pre‐adenylated DNA adapter (5′‐TGGAATTCTCGGGTGCCAAGG‐3′) was ligated to the 3′ end of the mRNA fragments at 4°C overnight using the T4 RNA ligase 2, truncated K227Q (New England Biolabs, Inc.), in 1× T4 RNA ligase buffer (no ATP) and 15% DMSO. .. After another round of RNeasy MinElute Cleanup Kit (Qiagen, Inc.), a pre‐adenylated DNA adapter (5′‐TGGAATTCTCGGGTGCCAAGG‐3′) was ligated to the 3′ end of the mRNA fragments at 4°C overnight using the T4 RNA ligase 2, truncated K227Q (New England Biolabs, Inc.), in 1× T4 RNA ligase buffer (no ATP) and 15% DMSO.

    Article Title: Histone gene replacement reveals a post-transcriptional role for H3K36 in maintaining metazoan transcriptome fidelity
    Article Snippet: For semi-quantitative PCR, PCR reactions were prepared in biological triplicate using 2x Red Master Mix (Apex Bioscience), and targets were amplified for 35 cycles of PCR with a 95°C denaturation step, a 60°C annealing step, and a 72°C elongation step. .. For LM-PAT, 1 µg total RNA was incubated with 5 pmol preadenylated lmPAT anchor primer (ppApCAGCTGTAGCTATGCGCACCGAGTCAGATCAG) (adenylated using 5’ DNA Adenylation Kit, NEB), and ligated with T4 RNA Ligase 2, truncated K227Q (NEB) using manufacturers protocol.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Preparation of Multiplexed Small RNA Libraries From Plants
    Article Snippet: Fiber glass (Corning Incorporated, catalog number: 988-10144) Extra thick blot paper (Bio-Rad Laboratories, catalog number: 170-3969) Whatman DE81 ion exchange cellulose chromatography paper (Thermo Fisher Scientific, catalog number: 05-717-1A) GlycoBlue coprecipitant (Life Technologies, catalog number: AM9516) 100% ethanol 75% ethanol EB buffer (QIAGEN, catalog number: 19086) Qubit RNA HS Assay Kit for quantification of purified small RNAs (Life Technologies, catalog number: ) Qubit dsDNA HS Assay Kit for quantification of DNA amplicon (Life Technologies, catalog number: ) Low salt buffer (see Recipes) High salt buffer (see Recipes) .. 8-Strip PCR thin-walled, 200 μl tubes (Corning Incorporated, Axygen® , catalog number: PCR-0208-CP-C) miRNA cloning linker 1 (/5′App/CTGTAGGCACCATCAAT/3′ddC/; 1 nm) (Integrated DNA Technologies, catalog number: 11-04-03-05) Truncated, K227Q mutation T4 RNA Ligase 2 (New England Biolabs, catalog number: M0351L) De-adenylase (New England Biolabs, catalog number: M0331S) Exonuclease VII (United State Biological, catalog number: 70082Z) RNA 5′ Adapter (GUUCAGAGUUCUACAGUCCGACGAUC) (Illumina RA5, catalog number: 15013205) dATP (Life Technologies, catalog number: 55082) T4 RNA Ligase I (Life Technologies, catalog number: AM2141) RT-PCR Primer (ATTGATGGTGCCTACAG; 25 nmol; de-salted) (Integrated DNA Technologies) SuperScript III (Life Technologies, catalog number: 18080051) Phusion High Fidelity II (Thermo Fisher Scientific, catalog number: F549L) 5′ PCR Primer (Illumina small RNA PCR primer 2: AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA) 3′ Indexed PCR Primer I1 – I12 (100 nmol; PAGE-purified) (Integrated DNA Technologies) Note: The barcodes below (underlined sequences) are reverse complemented in the final Illumina output sequence (see sequence in square brackets for each primer). .. I1 [CGATGT]: CAAGCAGAAGACGGCATACGA ACATCG ATTGATGGTGCCTACAG I2 [GATCAC]: CAAGCAGAAGACGGCATACGA GTGATC ATTGATGGTGCCTACAG I3 [CAGATG]: CAAGCAGAAGACGGCATACGA CATCTG ATTGATGGTGCCTACAG I4 [TACGTT]: CAAGCAGAAGACGGCATACGA AACGTA ATTGATGGTGCCTACAG I5 [TTACCA]: CAAGCAGAAGACGGCATACGA TGGTAA ATTGATGGTGCCTACAG I6 [ACTGTA]: CAAGCAGAAGACGGCATACGA TACAGT ATTGATGGTGCCTACAG I7 [ATCACG]: CAAGCAGAAGACGGCATACGA CGTGAT ATTGATGGTGCCTACAG I8 [ACTTGT]: CAAGCAGAAGACGGCATACGA ACAAGT ATTGATGGTGCCTACAG I9 [GCCAAT]: CAAGCAGAAGACGGCATACGA ATTGGC ATTGATGGTGCCTACAG I10 [TGCTAG]: CAAGCAGAAGACGGCATACGA CTAGCA ATTGATGGTGCCTACAG I11 [CTTGTA]: CAAGCAGAAGACGGCATACGA TACAAG ATTGATGGTGCCTACAG I12 [TCAGGC]: CAAGCAGAAGACGGCATACGA GCCTGA ATTGATGGTGCCTACAG QIAquick PCR Purification Kit (QIAGEN, catalog number: 28106) Diethylpyrocarbonate (DEPC)-H2 O/ RNase-free H2 O DNA size marker suitable for detecting a ~120 bp fragment (50 bp step ladder, Promega Corporation, catalog number: G4521; 25 bp step ladder, Promega Corporation, catalog number: G4511) 3 M sodium acetate (NaOAc) (pH 5.5) Reagents for 6% native PAGE 37.5: 1 polyacrylamide:bisacrylamide (see Recipes) TEMED (Life Technologies, catalog number: 15524-010) 10% Ammonium persulfate (Sigma, catalog number: A3678-100G) (See Recipes)

    Article Title: Transgenerational function of Tetrahymena Piwi protein Twi8p at distinctive noncoding RNA loci
    Article Snippet: Size selection of purified sRNAs was performed by denaturing PAGE. sRNAs were prepared for Solexa sequencing as previously described ( ). .. Briefly, IDT miRNA cloning linker 1 (Modban) was ligated to the 3′ end of the purified sRNAs using T4 RNA ligase, truncated, K227Q (NEB).

    Synthesized:

    Article Title: Biased transcription and selective degradation of small RNAs shape the pattern of DNA elimination in Tetrahymena
    Article Snippet: A 20-μL aliquot of the RNA was end-repaired and resuspended in 10.5 μL of water. cDNA was synthesized as described above in the section “Small RNA cDNA Library Preparation and Sequencing” with some modifications. .. For 3′ adapter ligation, 10 μL of the end-repaired RNAs was incubated with 1 μL of 50 μM 3′ adapter, 0.5 μL of Ribolock (Fermentas), 2.15 μL of ATP-free T4 RNA ligase buffer, 6 μL of 50% PEG-8000, and 2 μL of T4 RNA ligase 2-truncated K227Q (New England Biolabs) overnight at 16°C.

    Article Title: The Gcn4 transcription factor reduces protein synthesis capacity and extends yeast lifespan
    Article Snippet: Ligation of RNA 3′ adapter (RA3, part # 15013207, Illumina) was done using T4 RNA Ligase 2, truncated K227Q (M0351L, New England Biolabs Inc) according to the Illumina protocol. .. The RNA 5′ adapter (RA5, part #15013205, Illumina) was ligated using T4 RNA ligase (EL0021, Fermentas) according to the Illumina protocol, and the RNA was then purified to remove unligated 5′ adapters.

    Article Title: Nucleotide resolution mapping of influenza A virus nucleoprotein-RNA interactions reveals RNA features required for replication
    Article Snippet: 3′-OH RNA previously prepared by CIP treatment was ligated to a 3′ adaptor using T4 RNA Ligase 2, truncated K227Q (NEB), according to manufacturer’s instructions. .. Following isolation of adaptor ligated and radiolabeled RNA, all subsequent library generation steps were identical.

    Autoradiography:

    Article Title: Nucleotide resolution mapping of influenza A virus nucleoprotein-RNA interactions reveals RNA features required for replication
    Article Snippet: 32 P-labeled RNA was identified by autoradiography, excised, and extracted following proteinase K (New England Biolabs (NEB)) digestion. .. 3′-OH RNA previously prepared by CIP treatment was ligated to a 3′ adaptor using T4 RNA Ligase 2, truncated K227Q (NEB), according to manufacturer’s instructions.

    Blocking Assay:

    Article Title: BioTAP-XL - crosslinking/tandem affinity purification to study DNA targets, RNA and protein components of chromatin associated complexes
    Article Snippet: CRAC-TN150 buffer (see recipe) CRAC-Wash buffer 1 (see recipe) 1 x CRAC-PNK buffer (see recipe) 5 x CRAC-PNK buffer (see recipe) TruSeq ChIP Sample Prep Kit for ChIP-Seq (Illumina, IP-202-1012) RNace-It Ribonuclease Cocktail 6000U (Agilent Technologies Inc, cat. no. 400720) TSAP Thermosensitive Alkaline Phosphatase (Promega, cat. no. M9910) RNasin Recombinant Ribonuclease Inhibitor (Promega, cat. no. N2515) 10mM dNTP Mix (Invitrogen, cat. no. 18427-088) SuperScript III First-Strand Synthesis kit (Invitrogen, cat. no. 18080-051) Phusion Hot Start II DNA Polymerase 2 U/μL (Thermo Scientific, cat. no. F-549S) T4 RNA ligase 2 truncated K227Q and 50% PEG 8000 (New England BioLabs Inc, cat. no. M0351L) T4 RNA Ligase 1 (New England BioLabs Inc, cat. no. M0204L) T4 PNK-Polynucleotide Kinase from T4-infected Escherichia coli (Sigma, cat. no. P4390-500UN). .. CRAC-TN150 buffer (see recipe) CRAC-Wash buffer 1 (see recipe) 1 x CRAC-PNK buffer (see recipe) 5 x CRAC-PNK buffer (see recipe) TruSeq ChIP Sample Prep Kit for ChIP-Seq (Illumina, IP-202-1012) RNace-It Ribonuclease Cocktail 6000U (Agilent Technologies Inc, cat. no. 400720) TSAP Thermosensitive Alkaline Phosphatase (Promega, cat. no. M9910) RNasin Recombinant Ribonuclease Inhibitor (Promega, cat. no. N2515) 10mM dNTP Mix (Invitrogen, cat. no. 18427-088) SuperScript III First-Strand Synthesis kit (Invitrogen, cat. no. 18080-051) Phusion Hot Start II DNA Polymerase 2 U/μL (Thermo Scientific, cat. no. F-549S) T4 RNA ligase 2 truncated K227Q and 50% PEG 8000 (New England BioLabs Inc, cat. no. M0351L) T4 RNA Ligase 1 (New England BioLabs Inc, cat. no. M0204L) T4 PNK-Polynucleotide Kinase from T4-infected Escherichia coli (Sigma, cat. no. P4390-500UN).

    Concentration Assay:

    Article Title: Kinetic CRAC uncovers a role for Nab3 in determining gene expression profiles during stress
    Article Snippet: Beads were subsequently incubated with 80 µl of 1xPNK buffer containing eight units of TSAP alkaline phosphatase (Promega) and 80 units of recombinant RNasin (Promega) for 1 h at 37 °C. .. After one 500 µl wash with wash buffer I and three 500 µl washes with 1xPNK buffer, the beads were resuspended in 80 µl of 3′ linker ligation mix (1xPNK buffer, App-PE 3′ adapter (see Supplementary Table ; 0.6 µM final concentration), 10% PEG8000, 30 units of T4 RNA ligase 2 truncated K227Q (NEB), 60 units RNAsin (Promega)). .. The samples were incubated at 25 °C for 4–6 h. Following one 500 µl wash buffer I wash and three 1xPNK buffer washes, the beads were incubated with 60 µl of 5′end labeling mix (1xPNK buffer, 30µCi 32 P-γATP (Perkin Elmer) and 30 units of T4 polynucleotide kinase (NEB)) for 40 min at 37 °C.

    Article Title: 3′ end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character—RNA-Seq analyses
    Article Snippet: The 3′ ends of dephosphorylated RNAs were then ligated to 5′ pre-adenylated 3′ adapter with 3′ Biotin modification (the 3′ adapter sequence is shown in ), which was attached to Streptavidin Magnetic Beads (New England BioLabs) , by T4 RNA Ligase 2 truncated K227Q (New England BioLabs) ( ). .. After 3′ ligation, we performed three magnetic bead washing cycles to remove all unligated RNAs and NTPs.

    Real-time Polymerase Chain Reaction:

    Article Title: Histone gene replacement reveals a post-transcriptional role for H3K36 in maintaining metazoan transcriptome fidelity
    Article Snippet: For RT-qPCR, reactions were prepared in biological triplicate using Maxima SYBR Green/ROX qPCR Master Mix (Thermo Scientific), and fluorescence was monitored across 40 cycles in 96 well plate format. .. For LM-PAT, 1 µg total RNA was incubated with 5 pmol preadenylated lmPAT anchor primer (ppApCAGCTGTAGCTATGCGCACCGAGTCAGATCAG) (adenylated using 5’ DNA Adenylation Kit, NEB), and ligated with T4 RNA Ligase 2, truncated K227Q (NEB) using manufacturers protocol.

    cDNA Library Assay:

    Article Title: Biased transcription and selective degradation of small RNAs shape the pattern of DNA elimination in Tetrahymena
    Article Snippet: A 20-μL aliquot of the RNA was end-repaired and resuspended in 10.5 μL of water. cDNA was synthesized as described above in the section “Small RNA cDNA Library Preparation and Sequencing” with some modifications. .. For 3′ adapter ligation, 10 μL of the end-repaired RNAs was incubated with 1 μL of 50 μM 3′ adapter, 0.5 μL of Ribolock (Fermentas), 2.15 μL of ATP-free T4 RNA ligase buffer, 6 μL of 50% PEG-8000, and 2 μL of T4 RNA ligase 2-truncated K227Q (New England Biolabs) overnight at 16°C.

    Article Title: Substantial and robust changes in microRNA transcriptome support postnatal development of the hypothalamus in rat
    Article Snippet: Paragraph title: cDNA library construction ... To prevent secondary structures, mix were heated for 3 min at 70 °C, then kept on ice while adding [50%] PEG 8000 (2.4 ul), 80 U of truncated T4 RNA ligase 2 K227Q (0.4 ul) and the corresponding buffer (1 ul) (all provided by NEB).

    Incubation:

    Article Title: Kinetic CRAC uncovers a role for Nab3 in determining gene expression profiles during stress
    Article Snippet: Beads were subsequently incubated with 80 µl of 1xPNK buffer containing eight units of TSAP alkaline phosphatase (Promega) and 80 units of recombinant RNasin (Promega) for 1 h at 37 °C. .. After one 500 µl wash with wash buffer I and three 500 µl washes with 1xPNK buffer, the beads were resuspended in 80 µl of 3′ linker ligation mix (1xPNK buffer, App-PE 3′ adapter (see Supplementary Table ; 0.6 µM final concentration), 10% PEG8000, 30 units of T4 RNA ligase 2 truncated K227Q (NEB), 60 units RNAsin (Promega)).

    Article Title: Biased transcription and selective degradation of small RNAs shape the pattern of DNA elimination in Tetrahymena
    Article Snippet: A 20-μL aliquot of the RNA was end-repaired and resuspended in 10.5 μL of water. cDNA was synthesized as described above in the section “Small RNA cDNA Library Preparation and Sequencing” with some modifications. .. For 3′ adapter ligation, 10 μL of the end-repaired RNAs was incubated with 1 μL of 50 μM 3′ adapter, 0.5 μL of Ribolock (Fermentas), 2.15 μL of ATP-free T4 RNA ligase buffer, 6 μL of 50% PEG-8000, and 2 μL of T4 RNA ligase 2-truncated K227Q (New England Biolabs) overnight at 16°C. .. The RNAs were enriched on anti-BrdU beads as above and resuspended in 10 μL of water.

    Article Title: 3′ end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character—RNA-Seq analyses
    Article Snippet: Transcripts were then incubated with Shrimp Alkaline Phosphatase/rSAP (New England BioLabs) at 37°C for 45 min to dephosphorylate the 5′ end of RNAs and to hydrolyze remaining NTPs from the reaction. .. The 3′ ends of dephosphorylated RNAs were then ligated to 5′ pre-adenylated 3′ adapter with 3′ Biotin modification (the 3′ adapter sequence is shown in ), which was attached to Streptavidin Magnetic Beads (New England BioLabs) , by T4 RNA Ligase 2 truncated K227Q (New England BioLabs) ( ).

    Article Title: A Novel Epigenetic Silencing Pathway Involving the Highly Conserved 5’-3’ Exoribonuclease Dhp1/Rat1/Xrn2 in Schizosaccharomyces pombe
    Article Snippet: Samples were incubated with 50 μl of nickel agarose beads (Macherey-Nagel) over night at 4°C. .. All washes, alkaline phosphatase treatment and 3’ linker ligation were carried out as described except that 40U T4 RNA ligase 2 truncated K227Q (NEB) was used instead of T4 RNA ligase.

    Article Title: The Gcn4 transcription factor reduces protein synthesis capacity and extends yeast lifespan
    Article Snippet: The fragmented mRNA was further incubated with ATP and T4 polynucleotide kinase (EK0032, Fermentas) at 37 °C for 1 h and subsequently purified. .. Ligation of RNA 3′ adapter (RA3, part # 15013207, Illumina) was done using T4 RNA Ligase 2, truncated K227Q (M0351L, New England Biolabs Inc) according to the Illumina protocol.

    Article Title: Substantial and robust changes in microRNA transcriptome support postnatal development of the hypothalamus in rat
    Article Snippet: 3′-Adaptor (25 pmoles) was adenylated as described using 1600 U of T4 DNA ligase (NEB) and the Quick Ligation reaction buffer (NEB) in a volume of 50 ul by overnight incubation at 37 °C. .. To prevent secondary structures, mix were heated for 3 min at 70 °C, then kept on ice while adding [50%] PEG 8000 (2.4 ul), 80 U of truncated T4 RNA ligase 2 K227Q (0.4 ul) and the corresponding buffer (1 ul) (all provided by NEB).

    Article Title: Global analysis of CPSF2-mediated alternative splicing: Integration of global iCLIP and transcriptome profiling data
    Article Snippet: Samples were then washed twice with high-salt wash buffer and once with 900 μl PNK wash buffer. .. To ligate linkers to the 3′ end of RNAs, 4.5 μl of purified RNA was mixed with 1 μl of 10 μM miRCat33 Adaptor (Integrated DNA Technologies), 1.5 μl of RNA Ligase buffer, 0.5 μl of T4 RNA Ligase 2, Truncated K227Q (NEB), and 7.5 μl of PEG 8000, and incubated at 30 °C for 6 h. Samples were then end-labeled using PNK enzyme and run on a NuPAGE Novex 10% Bis–Tris Protein Gel (Invitrogen) with 1 × MOPS running buffer (Invitrogen). .. Proteins and covalently bound RNAs were then transferred to a nitrocellulose membrane (Whatman) using a Novex wet transfer apparatus (Invitrogen).

    Article Title: Histone gene replacement reveals a post-transcriptional role for H3K36 in maintaining metazoan transcriptome fidelity
    Article Snippet: For RT-qPCR, reactions were prepared in biological triplicate using Maxima SYBR Green/ROX qPCR Master Mix (Thermo Scientific), and fluorescence was monitored across 40 cycles in 96 well plate format. .. For LM-PAT, 1 µg total RNA was incubated with 5 pmol preadenylated lmPAT anchor primer (ppApCAGCTGTAGCTATGCGCACCGAGTCAGATCAG) (adenylated using 5’ DNA Adenylation Kit, NEB), and ligated with T4 RNA Ligase 2, truncated K227Q (NEB) using manufacturers protocol. .. Ligated RNA was reverse-transcribed with Superscript III (Life Technologies) using an lmPAT RT primer (GACTCGGTGCGCATAGCTACAGCTG).

    Article Title: The microRNAs in an Ancient Protist Repress the Variant-Specific Surface Protein Expression by Targeting the Entire Coding Sequence
    Article Snippet: For the addition of a sonication step to the procedure, the cells were resuspend in RIPA buffer as described previously and sonicated twice with five 1 second bursts at a 20% duty cycle. .. The purified small RNAs were ligated at the 3′-end to 5–10 µM gel purified miRNA Cloning Linker-1 (IDT) using T4 RNA ligase 2 and truncated with K227Q (NEB) by incubating at 25°C for 1 hr, 15°C for 2 hrs and followed by a final 4°C incubation overnight. .. The reaction mixture was analyzed in 15% polyacrylamide/8 M Urea gel, and the band of ligated product was excised.

    Article Title: Transgenerational function of Tetrahymena Piwi protein Twi8p at distinctive noncoding RNA loci
    Article Snippet: Bound Twi8 RNPs were eluted by cleavage with Tobacco Etch Virus protease, and the eluate was incubated with Flag M2 antibody resin. .. Briefly, IDT miRNA cloning linker 1 (Modban) was ligated to the 3′ end of the purified sRNAs using T4 RNA ligase, truncated, K227Q (NEB).

    Activity Assay:

    Article Title: 3′ end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character—RNA-Seq analyses
    Article Snippet: The 3′ ends of dephosphorylated RNAs were then ligated to 5′ pre-adenylated 3′ adapter with 3′ Biotin modification (the 3′ adapter sequence is shown in ), which was attached to Streptavidin Magnetic Beads (New England BioLabs) , by T4 RNA Ligase 2 truncated K227Q (New England BioLabs) ( ). .. The 3′ ends of dephosphorylated RNAs were then ligated to 5′ pre-adenylated 3′ adapter with 3′ Biotin modification (the 3′ adapter sequence is shown in ), which was attached to Streptavidin Magnetic Beads (New England BioLabs) , by T4 RNA Ligase 2 truncated K227Q (New England BioLabs) ( ).

    Modification:

    Article Title: 3′ end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character—RNA-Seq analyses
    Article Snippet: Afterward, phosphatase activity was eliminated by heat inactivation at 65°C for 5 min. .. The 3′ ends of dephosphorylated RNAs were then ligated to 5′ pre-adenylated 3′ adapter with 3′ Biotin modification (the 3′ adapter sequence is shown in ), which was attached to Streptavidin Magnetic Beads (New England BioLabs) , by T4 RNA Ligase 2 truncated K227Q (New England BioLabs) ( ). .. The 40 μl ligation reactions containing 20% (w/v) PEG 8000, 0.05 mg/ml bovine serum albumin, 50 mM NaCl and 2 μl of RNA Ligase 2 at 200 U μl−1 were incubated at 16°C overnight.

    Article Title: The microRNAs in an Ancient Protist Repress the Variant-Specific Surface Protein Expression by Targeting the Entire Coding Sequence
    Article Snippet: The NGS libraries of GlAgo associated small RNAs were constructed using a modified version of the Mello Lab protocol ( www.lsi.umich.edu/files/SmallRNACloning.pdf ). .. The purified small RNAs were ligated at the 3′-end to 5–10 µM gel purified miRNA Cloning Linker-1 (IDT) using T4 RNA ligase 2 and truncated with K227Q (NEB) by incubating at 25°C for 1 hr, 15°C for 2 hrs and followed by a final 4°C incubation overnight.

    Western Blot:

    Article Title: The microRNAs in an Ancient Protist Repress the Variant-Specific Surface Protein Expression by Targeting the Entire Coding Sequence
    Article Snippet: Briefly, the GlAgo associated small RNAs were isolated from Giardia WB trophozoites as previously described and resuspended in 10 µl RNase-free diH2 O . .. The purified small RNAs were ligated at the 3′-end to 5–10 µM gel purified miRNA Cloning Linker-1 (IDT) using T4 RNA ligase 2 and truncated with K227Q (NEB) by incubating at 25°C for 1 hr, 15°C for 2 hrs and followed by a final 4°C incubation overnight.

    Ligation:

    Article Title: Kinetic CRAC uncovers a role for Nab3 in determining gene expression profiles during stress
    Article Snippet: Beads were subsequently incubated with 80 µl of 1xPNK buffer containing eight units of TSAP alkaline phosphatase (Promega) and 80 units of recombinant RNasin (Promega) for 1 h at 37 °C. .. After one 500 µl wash with wash buffer I and three 500 µl washes with 1xPNK buffer, the beads were resuspended in 80 µl of 3′ linker ligation mix (1xPNK buffer, App-PE 3′ adapter (see Supplementary Table ; 0.6 µM final concentration), 10% PEG8000, 30 units of T4 RNA ligase 2 truncated K227Q (NEB), 60 units RNAsin (Promega)). .. The samples were incubated at 25 °C for 4–6 h. Following one 500 µl wash buffer I wash and three 1xPNK buffer washes, the beads were incubated with 60 µl of 5′end labeling mix (1xPNK buffer, 30µCi 32 P-γATP (Perkin Elmer) and 30 units of T4 polynucleotide kinase (NEB)) for 40 min at 37 °C.

    Article Title: Biased transcription and selective degradation of small RNAs shape the pattern of DNA elimination in Tetrahymena
    Article Snippet: A 20-μL aliquot of the RNA was end-repaired and resuspended in 10.5 μL of water. cDNA was synthesized as described above in the section “Small RNA cDNA Library Preparation and Sequencing” with some modifications. .. For 3′ adapter ligation, 10 μL of the end-repaired RNAs was incubated with 1 μL of 50 μM 3′ adapter, 0.5 μL of Ribolock (Fermentas), 2.15 μL of ATP-free T4 RNA ligase buffer, 6 μL of 50% PEG-8000, and 2 μL of T4 RNA ligase 2-truncated K227Q (New England Biolabs) overnight at 16°C. .. The RNAs were enriched on anti-BrdU beads as above and resuspended in 10 μL of water.

    Article Title: 3′ end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character—RNA-Seq analyses
    Article Snippet: The 3′ ends of dephosphorylated RNAs were then ligated to 5′ pre-adenylated 3′ adapter with 3′ Biotin modification (the 3′ adapter sequence is shown in ), which was attached to Streptavidin Magnetic Beads (New England BioLabs) , by T4 RNA Ligase 2 truncated K227Q (New England BioLabs) ( ). .. The 40 μl ligation reactions containing 20% (w/v) PEG 8000, 0.05 mg/ml bovine serum albumin, 50 mM NaCl and 2 μl of RNA Ligase 2 at 200 U μl−1 were incubated at 16°C overnight.

    Article Title: A Novel Epigenetic Silencing Pathway Involving the Highly Conserved 5’-3’ Exoribonuclease Dhp1/Rat1/Xrn2 in Schizosaccharomyces pombe
    Article Snippet: Samples were incubated with 50 μl of nickel agarose beads (Macherey-Nagel) over night at 4°C. .. All washes, alkaline phosphatase treatment and 3’ linker ligation were carried out as described except that 40U T4 RNA ligase 2 truncated K227Q (NEB) was used instead of T4 RNA ligase. .. The beads were incubated in 80 μl phosphorylation mix (16 μl 5x PNK buffer (250 mM Tris-HCl (pH 7.8), 50 mM MgCl2 , 50 mM β-mercaptoethanol), 200 mM ATP (Sigma, A6559), 20U T4 polynucleotide kinase (NEB), 80U RNase Inhibitor) for 40 min at 37°C.

    Article Title: The Gcn4 transcription factor reduces protein synthesis capacity and extends yeast lifespan
    Article Snippet: The fragmented mRNA was further incubated with ATP and T4 polynucleotide kinase (EK0032, Fermentas) at 37 °C for 1 h and subsequently purified. .. Ligation of RNA 3′ adapter (RA3, part # 15013207, Illumina) was done using T4 RNA Ligase 2, truncated K227Q (M0351L, New England Biolabs Inc) according to the Illumina protocol. .. The ligation step was followed by RNA purification as described above to remove unligated 3′ adapters.

    Article Title: BioTAP-XL - crosslinking/tandem affinity purification to study DNA targets, RNA and protein components of chromatin associated complexes
    Article Snippet: CRAC-TN150 buffer (see recipe) CRAC-Wash buffer 1 (see recipe) 1 x CRAC-PNK buffer (see recipe) 5 x CRAC-PNK buffer (see recipe) TruSeq ChIP Sample Prep Kit for ChIP-Seq (Illumina, IP-202-1012) RNace-It Ribonuclease Cocktail 6000U (Agilent Technologies Inc, cat. no. 400720) TSAP Thermosensitive Alkaline Phosphatase (Promega, cat. no. M9910) RNasin Recombinant Ribonuclease Inhibitor (Promega, cat. no. N2515) 10mM dNTP Mix (Invitrogen, cat. no. 18427-088) SuperScript III First-Strand Synthesis kit (Invitrogen, cat. no. 18080-051) Phusion Hot Start II DNA Polymerase 2 U/μL (Thermo Scientific, cat. no. F-549S) T4 RNA ligase 2 truncated K227Q and 50% PEG 8000 (New England BioLabs Inc, cat. no. M0351L) T4 RNA Ligase 1 (New England BioLabs Inc, cat. no. M0204L) T4 PNK-Polynucleotide Kinase from T4-infected Escherichia coli (Sigma, cat. no. P4390-500UN). .. CRAC-TN150 buffer (see recipe) CRAC-Wash buffer 1 (see recipe) 1 x CRAC-PNK buffer (see recipe) 5 x CRAC-PNK buffer (see recipe) TruSeq ChIP Sample Prep Kit for ChIP-Seq (Illumina, IP-202-1012) RNace-It Ribonuclease Cocktail 6000U (Agilent Technologies Inc, cat. no. 400720) TSAP Thermosensitive Alkaline Phosphatase (Promega, cat. no. M9910) RNasin Recombinant Ribonuclease Inhibitor (Promega, cat. no. N2515) 10mM dNTP Mix (Invitrogen, cat. no. 18427-088) SuperScript III First-Strand Synthesis kit (Invitrogen, cat. no. 18080-051) Phusion Hot Start II DNA Polymerase 2 U/μL (Thermo Scientific, cat. no. F-549S) T4 RNA ligase 2 truncated K227Q and 50% PEG 8000 (New England BioLabs Inc, cat. no. M0351L) T4 RNA Ligase 1 (New England BioLabs Inc, cat. no. M0204L) T4 PNK-Polynucleotide Kinase from T4-infected Escherichia coli (Sigma, cat. no. P4390-500UN).

    Article Title: Oligoadenylation of 3′ decay intermediates promotes cytoplasmic mRNA degradation in Drosophila cells
    Article Snippet: In the first data set, a control ligation in the absence of ATP was carried out with a preadenylated adaptor and T4 RNA ligase 1. .. In the second data set, truncated T4 RNA Ligase 2 K227Q (NEB) was used for the same type of control.

    Article Title: Substantial and robust changes in microRNA transcriptome support postnatal development of the hypothalamus in rat
    Article Snippet: 3′-Adaptor (25 pmoles) was adenylated as described using 1600 U of T4 DNA ligase (NEB) and the Quick Ligation reaction buffer (NEB) in a volume of 50 ul by overnight incubation at 37 °C. .. To prevent secondary structures, mix were heated for 3 min at 70 °C, then kept on ice while adding [50%] PEG 8000 (2.4 ul), 80 U of truncated T4 RNA ligase 2 K227Q (0.4 ul) and the corresponding buffer (1 ul) (all provided by NEB).

    Article Title: Evaluating bias-reducing protocols for RNA sequencing library preparation
    Article Snippet: For the CircLig libraries, 40 ng RNA pool (~6 pmol) and 100 ng Universal Cloning Linker (~18 pmol; NEB) were denatured at 80°C for 2 min then placed immediately on ice. .. Ligation with 200 U T4 RNA Ligase 2 truncated K227Q mutant (trRnl2 K227Q; NEB) was performed in the presence of 1X RNA ligase buffer (NEB), 15% PEG8000, 20 U SuperaseIn (Ambion), at 25°C for 3 hr. .. Ligation with 20 pmol Mth K97A was performed in 1X NEB1 buffer and 20 U SuperaseIn, at 65°C for 1 hr, as described by Zhelkovsky and McReynolds (11).

    Article Title: The ribonuclease activity of SAMHD1 is required for HIV-1 restriction
    Article Snippet: 35–60 nucleotide RNA was extracted in a polyacrylamide, 7 M urea, TBE gel. .. 3′ adaptor (5′/rApp/TGGAATTCTCGGGTGCCAAGG/ddC/-3′, IDT) was ligated using T4 RNA ligase truncated K227Q (NEB) followed by ligation of 5′ adaptor (5′ Solexa linker, 100-M; 5′rGrUrUrCrArGrArGrUrUrCrUrArCrArGrUrCrCrGrArCrGrA rUrC-3′, IDT) using T4 RNA ligase (TaKaRa). .. The 5′ and 3′ adaptor-ligated RNA was reverse transcribed using RNA RT Primer (RTP; 5′GCCTTGGCACCCGAGAATTCCA-3′, IDT) and SuperScript™ III reverse transcriptase (Invitrogen).

    Article Title: Elimination of Ligation Dependent Artifacts in T4 RNA Ligase to Achieve High Efficiency and Low Bias MicroRNA Capture
    Article Snippet: Paragraph title: Ligation Protocol ... In the experiments where different ligases were investigated, T4 RNA Ligase 2 truncated, T4 RNA Ligase 2 truncated R55K K227Q, and Thermostable 5′ App DNA/RNA Ligase were all obtained from New England Biolabs.

    Article Title: Nucleotide resolution mapping of influenza A virus nucleoprotein-RNA interactions reveals RNA features required for replication
    Article Snippet: To account for potential bias introduced during adaptor ligation and sequencing, we also generated RNA-seq libraries using RNA extracted from uncross-linked cell lysates of influenza infected 293T cells from which RNA was isolated by TRIzol (Invitrogen) extraction, then 10 μg RNA was fragmented by Mg2+ at 95 °C for 12 min (NEB, Magnesium RNA Fragmentation Module). .. 3′-OH RNA previously prepared by CIP treatment was ligated to a 3′ adaptor using T4 RNA Ligase 2, truncated K227Q (NEB), according to manufacturer’s instructions.

    Infection:

    Article Title: The ribonuclease activity of SAMHD1 is required for HIV-1 restriction
    Article Snippet: Total RNA was extracted from mock- and SAMHD1-expressing U937 cells uninfected or infected with HIV-1 using TRIzol reagent (Invitrogen) by manufacturer's instruction followed by addition of spike-in poly(A)-tailed RNA (GeneChip Eukaryotic Poly-A RNA control Kit, Affymetrix) to reduce background noise and allow comparison between samples. .. 3′ adaptor (5′/rApp/TGGAATTCTCGGGTGCCAAGG/ddC/-3′, IDT) was ligated using T4 RNA ligase truncated K227Q (NEB) followed by ligation of 5′ adaptor (5′ Solexa linker, 100-M; 5′rGrUrUrCrArGrArGrUrUrCrUrArCrArGrUrCrCrGrArCrGrA rUrC-3′, IDT) using T4 RNA ligase (TaKaRa).

    Article Title: Nucleotide resolution mapping of influenza A virus nucleoprotein-RNA interactions reveals RNA features required for replication
    Article Snippet: To account for potential bias introduced during adaptor ligation and sequencing, we also generated RNA-seq libraries using RNA extracted from uncross-linked cell lysates of influenza infected 293T cells from which RNA was isolated by TRIzol (Invitrogen) extraction, then 10 μg RNA was fragmented by Mg2+ at 95 °C for 12 min (NEB, Magnesium RNA Fragmentation Module). .. 3′-OH RNA previously prepared by CIP treatment was ligated to a 3′ adaptor using T4 RNA Ligase 2, truncated K227Q (NEB), according to manufacturer’s instructions.

    Generated:

    Article Title: Oligoadenylation of 3′ decay intermediates promotes cytoplasmic mRNA degradation in Drosophila cells
    Article Snippet: In the second data set, truncated T4 RNA Ligase 2 K227Q (NEB) was used for the same type of control. .. After G50 gel filtration, phenol–chloroform extraction and ethanol precipitation, ligation products were reverse-transcribed (M-MLV reverse transcriptase, Promega) with an adaptor reverse oligonucleotide.

    Article Title: Nucleotide resolution mapping of influenza A virus nucleoprotein-RNA interactions reveals RNA features required for replication
    Article Snippet: To account for potential bias introduced during adaptor ligation and sequencing, we also generated RNA-seq libraries using RNA extracted from uncross-linked cell lysates of influenza infected 293T cells from which RNA was isolated by TRIzol (Invitrogen) extraction, then 10 μg RNA was fragmented by Mg2+ at 95 °C for 12 min (NEB, Magnesium RNA Fragmentation Module). .. 3′-OH RNA previously prepared by CIP treatment was ligated to a 3′ adaptor using T4 RNA Ligase 2, truncated K227Q (NEB), according to manufacturer’s instructions.

    Polymerase Chain Reaction:

    Article Title: The Gcn4 transcription factor reduces protein synthesis capacity and extends yeast lifespan
    Article Snippet: Ligation of RNA 3′ adapter (RA3, part # 15013207, Illumina) was done using T4 RNA Ligase 2, truncated K227Q (M0351L, New England Biolabs Inc) according to the Illumina protocol. .. Ligation of RNA 3′ adapter (RA3, part # 15013207, Illumina) was done using T4 RNA Ligase 2, truncated K227Q (M0351L, New England Biolabs Inc) according to the Illumina protocol.

    Article Title: Preparation of Multiplexed Small RNA Libraries From Plants
    Article Snippet: Fiber glass (Corning Incorporated, catalog number: 988-10144) Extra thick blot paper (Bio-Rad Laboratories, catalog number: 170-3969) Whatman DE81 ion exchange cellulose chromatography paper (Thermo Fisher Scientific, catalog number: 05-717-1A) GlycoBlue coprecipitant (Life Technologies, catalog number: AM9516) 100% ethanol 75% ethanol EB buffer (QIAGEN, catalog number: 19086) Qubit RNA HS Assay Kit for quantification of purified small RNAs (Life Technologies, catalog number: ) Qubit dsDNA HS Assay Kit for quantification of DNA amplicon (Life Technologies, catalog number: ) Low salt buffer (see Recipes) High salt buffer (see Recipes) .. 8-Strip PCR thin-walled, 200 μl tubes (Corning Incorporated, Axygen® , catalog number: PCR-0208-CP-C) miRNA cloning linker 1 (/5′App/CTGTAGGCACCATCAAT/3′ddC/; 1 nm) (Integrated DNA Technologies, catalog number: 11-04-03-05) Truncated, K227Q mutation T4 RNA Ligase 2 (New England Biolabs, catalog number: M0351L) De-adenylase (New England Biolabs, catalog number: M0331S) Exonuclease VII (United State Biological, catalog number: 70082Z) RNA 5′ Adapter (GUUCAGAGUUCUACAGUCCGACGAUC) (Illumina RA5, catalog number: 15013205) dATP (Life Technologies, catalog number: 55082) T4 RNA Ligase I (Life Technologies, catalog number: AM2141) RT-PCR Primer (ATTGATGGTGCCTACAG; 25 nmol; de-salted) (Integrated DNA Technologies) SuperScript III (Life Technologies, catalog number: 18080051) Phusion High Fidelity II (Thermo Fisher Scientific, catalog number: F549L) 5′ PCR Primer (Illumina small RNA PCR primer 2: AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA) 3′ Indexed PCR Primer I1 – I12 (100 nmol; PAGE-purified) (Integrated DNA Technologies) Note: The barcodes below (underlined sequences) are reverse complemented in the final Illumina output sequence (see sequence in square brackets for each primer). .. I1 [CGATGT]: CAAGCAGAAGACGGCATACGA ACATCG ATTGATGGTGCCTACAG I2 [GATCAC]: CAAGCAGAAGACGGCATACGA GTGATC ATTGATGGTGCCTACAG I3 [CAGATG]: CAAGCAGAAGACGGCATACGA CATCTG ATTGATGGTGCCTACAG I4 [TACGTT]: CAAGCAGAAGACGGCATACGA AACGTA ATTGATGGTGCCTACAG I5 [TTACCA]: CAAGCAGAAGACGGCATACGA TGGTAA ATTGATGGTGCCTACAG I6 [ACTGTA]: CAAGCAGAAGACGGCATACGA TACAGT ATTGATGGTGCCTACAG I7 [ATCACG]: CAAGCAGAAGACGGCATACGA CGTGAT ATTGATGGTGCCTACAG I8 [ACTTGT]: CAAGCAGAAGACGGCATACGA ACAAGT ATTGATGGTGCCTACAG I9 [GCCAAT]: CAAGCAGAAGACGGCATACGA ATTGGC ATTGATGGTGCCTACAG I10 [TGCTAG]: CAAGCAGAAGACGGCATACGA CTAGCA ATTGATGGTGCCTACAG I11 [CTTGTA]: CAAGCAGAAGACGGCATACGA TACAAG ATTGATGGTGCCTACAG I12 [TCAGGC]: CAAGCAGAAGACGGCATACGA GCCTGA ATTGATGGTGCCTACAG QIAquick PCR Purification Kit (QIAGEN, catalog number: 28106) Diethylpyrocarbonate (DEPC)-H2 O/ RNase-free H2 O DNA size marker suitable for detecting a ~120 bp fragment (50 bp step ladder, Promega Corporation, catalog number: G4521; 25 bp step ladder, Promega Corporation, catalog number: G4511) 3 M sodium acetate (NaOAc) (pH 5.5) Reagents for 6% native PAGE 37.5: 1 polyacrylamide:bisacrylamide (see Recipes) TEMED (Life Technologies, catalog number: 15524-010) 10% Ammonium persulfate (Sigma, catalog number: A3678-100G) (See Recipes)

    Article Title: The Coding Regions of Germline mRNAs Confer Sensitivity to Argonaute Regulation in C. elegans
    Article Snippet: Samples from wild-type and mutant were pretreated with a homemade 5′ polyphosphatase and were ligated to the 3′ adaptor linker 1 (5′ rAppAGATCGGAAGAGCACACGTCTGAACTCCAGTCA/3ddC/3′, IDT) using T4 RNA ligase 2 (M0351S, NEB). .. Samples from wild-type and mutant were pretreated with a homemade 5′ polyphosphatase and were ligated to the 3′ adaptor linker 1 (5′ rAppAGATCGGAAGAGCACACGTCTGAACTCCAGTCA/3ddC/3′, IDT) using T4 RNA ligase 2 (M0351S, NEB).

    Article Title: Oligoadenylation of 3′ decay intermediates promotes cytoplasmic mRNA degradation in Drosophila cells
    Article Snippet: In the second data set, truncated T4 RNA Ligase 2 K227Q (NEB) was used for the same type of control. .. Eight different forward primer combinations spanning the reporter RNA were used together with a reverse primer complementary to the 3′ ligation adaptor.

    Article Title: Substantial and robust changes in microRNA transcriptome support postnatal development of the hypothalamus in rat
    Article Snippet: Individual cDNA libraries were built following an Illumina-like protocol in which 3′- and 5′-adapters were sequentially ligated at the 3′ and 5′ ends of small RNAs, respectively, to allow for reverse transcription (RT) and amplification by polymerase chain reaction (PCR). .. To prevent secondary structures, mix were heated for 3 min at 70 °C, then kept on ice while adding [50%] PEG 8000 (2.4 ul), 80 U of truncated T4 RNA ligase 2 K227Q (0.4 ul) and the corresponding buffer (1 ul) (all provided by NEB).

    Article Title: The ribonuclease activity of SAMHD1 is required for HIV-1 restriction
    Article Snippet: 3′ adaptor (5′/rApp/TGGAATTCTCGGGTGCCAAGG/ddC/-3′, IDT) was ligated using T4 RNA ligase truncated K227Q (NEB) followed by ligation of 5′ adaptor (5′ Solexa linker, 100-M; 5′rGrUrUrCrArGrArGrUrUrCrUrArCrArGrUrCrCrGrArCrGrA rUrC-3′, IDT) using T4 RNA ligase (TaKaRa). .. 3′ adaptor (5′/rApp/TGGAATTCTCGGGTGCCAAGG/ddC/-3′, IDT) was ligated using T4 RNA ligase truncated K227Q (NEB) followed by ligation of 5′ adaptor (5′ Solexa linker, 100-M; 5′rGrUrUrCrArGrArGrUrUrCrUrArCrArGrUrCrCrGrArCrGrA rUrC-3′, IDT) using T4 RNA ligase (TaKaRa).

    Article Title: Single‐cell mRNA profiling reveals the hierarchical response of mi RNA targets to mi RNA induction
    Article Snippet: After another round of RNeasy MinElute Cleanup Kit (Qiagen, Inc.), a pre‐adenylated DNA adapter (5′‐TGGAATTCTCGGGTGCCAAGG‐3′) was ligated to the 3′ end of the mRNA fragments at 4°C overnight using the T4 RNA ligase 2, truncated K227Q (New England Biolabs, Inc.), in 1× T4 RNA ligase buffer (no ATP) and 15% DMSO. .. After another round of RNeasy MinElute Cleanup Kit (Qiagen, Inc.), a pre‐adenylated DNA adapter (5′‐TGGAATTCTCGGGTGCCAAGG‐3′) was ligated to the 3′ end of the mRNA fragments at 4°C overnight using the T4 RNA ligase 2, truncated K227Q (New England Biolabs, Inc.), in 1× T4 RNA ligase buffer (no ATP) and 15% DMSO.

    Article Title: Histone gene replacement reveals a post-transcriptional role for H3K36 in maintaining metazoan transcriptome fidelity
    Article Snippet: Paragraph title: Reverse transcription and PCR assays ... For LM-PAT, 1 µg total RNA was incubated with 5 pmol preadenylated lmPAT anchor primer (ppApCAGCTGTAGCTATGCGCACCGAGTCAGATCAG) (adenylated using 5’ DNA Adenylation Kit, NEB), and ligated with T4 RNA Ligase 2, truncated K227Q (NEB) using manufacturers protocol.

    Article Title: Transgenerational function of Tetrahymena Piwi protein Twi8p at distinctive noncoding RNA loci
    Article Snippet: Briefly, IDT miRNA cloning linker 1 (Modban) was ligated to the 3′ end of the purified sRNAs using T4 RNA ligase, truncated, K227Q (NEB). .. The sequencing adaptor was ligated to sRNA 5′ ends by T4 RNA ligase 1, and the intended products were gel-purified and collected by ethanol precipitation.

    Imaging:

    Article Title: The ribonuclease activity of SAMHD1 is required for HIV-1 restriction
    Article Snippet: 3′ adaptor (5′/rApp/TGGAATTCTCGGGTGCCAAGG/ddC/-3′, IDT) was ligated using T4 RNA ligase truncated K227Q (NEB) followed by ligation of 5′ adaptor (5′ Solexa linker, 100-M; 5′rGrUrUrCrArGrArGrUrUrCrUrArCrArGrUrCrCrGrArCrGrA rUrC-3′, IDT) using T4 RNA ligase (TaKaRa). .. RNA-Seq library was sequenced with 51-bp single-end sequencing in an Illumina HiSeq2500 sequencer.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Preparation of Multiplexed Small RNA Libraries From Plants
    Article Snippet: Fiber glass (Corning Incorporated, catalog number: 988-10144) Extra thick blot paper (Bio-Rad Laboratories, catalog number: 170-3969) Whatman DE81 ion exchange cellulose chromatography paper (Thermo Fisher Scientific, catalog number: 05-717-1A) GlycoBlue coprecipitant (Life Technologies, catalog number: AM9516) 100% ethanol 75% ethanol EB buffer (QIAGEN, catalog number: 19086) Qubit RNA HS Assay Kit for quantification of purified small RNAs (Life Technologies, catalog number: ) Qubit dsDNA HS Assay Kit for quantification of DNA amplicon (Life Technologies, catalog number: ) Low salt buffer (see Recipes) High salt buffer (see Recipes) .. 8-Strip PCR thin-walled, 200 μl tubes (Corning Incorporated, Axygen® , catalog number: PCR-0208-CP-C) miRNA cloning linker 1 (/5′App/CTGTAGGCACCATCAAT/3′ddC/; 1 nm) (Integrated DNA Technologies, catalog number: 11-04-03-05) Truncated, K227Q mutation T4 RNA Ligase 2 (New England Biolabs, catalog number: M0351L) De-adenylase (New England Biolabs, catalog number: M0331S) Exonuclease VII (United State Biological, catalog number: 70082Z) RNA 5′ Adapter (GUUCAGAGUUCUACAGUCCGACGAUC) (Illumina RA5, catalog number: 15013205) dATP (Life Technologies, catalog number: 55082) T4 RNA Ligase I (Life Technologies, catalog number: AM2141) RT-PCR Primer (ATTGATGGTGCCTACAG; 25 nmol; de-salted) (Integrated DNA Technologies) SuperScript III (Life Technologies, catalog number: 18080051) Phusion High Fidelity II (Thermo Fisher Scientific, catalog number: F549L) 5′ PCR Primer (Illumina small RNA PCR primer 2: AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA) 3′ Indexed PCR Primer I1 – I12 (100 nmol; PAGE-purified) (Integrated DNA Technologies) Note: The barcodes below (underlined sequences) are reverse complemented in the final Illumina output sequence (see sequence in square brackets for each primer). .. I1 [CGATGT]: CAAGCAGAAGACGGCATACGA ACATCG ATTGATGGTGCCTACAG I2 [GATCAC]: CAAGCAGAAGACGGCATACGA GTGATC ATTGATGGTGCCTACAG I3 [CAGATG]: CAAGCAGAAGACGGCATACGA CATCTG ATTGATGGTGCCTACAG I4 [TACGTT]: CAAGCAGAAGACGGCATACGA AACGTA ATTGATGGTGCCTACAG I5 [TTACCA]: CAAGCAGAAGACGGCATACGA TGGTAA ATTGATGGTGCCTACAG I6 [ACTGTA]: CAAGCAGAAGACGGCATACGA TACAGT ATTGATGGTGCCTACAG I7 [ATCACG]: CAAGCAGAAGACGGCATACGA CGTGAT ATTGATGGTGCCTACAG I8 [ACTTGT]: CAAGCAGAAGACGGCATACGA ACAAGT ATTGATGGTGCCTACAG I9 [GCCAAT]: CAAGCAGAAGACGGCATACGA ATTGGC ATTGATGGTGCCTACAG I10 [TGCTAG]: CAAGCAGAAGACGGCATACGA CTAGCA ATTGATGGTGCCTACAG I11 [CTTGTA]: CAAGCAGAAGACGGCATACGA TACAAG ATTGATGGTGCCTACAG I12 [TCAGGC]: CAAGCAGAAGACGGCATACGA GCCTGA ATTGATGGTGCCTACAG QIAquick PCR Purification Kit (QIAGEN, catalog number: 28106) Diethylpyrocarbonate (DEPC)-H2 O/ RNase-free H2 O DNA size marker suitable for detecting a ~120 bp fragment (50 bp step ladder, Promega Corporation, catalog number: G4521; 25 bp step ladder, Promega Corporation, catalog number: G4511) 3 M sodium acetate (NaOAc) (pH 5.5) Reagents for 6% native PAGE 37.5: 1 polyacrylamide:bisacrylamide (see Recipes) TEMED (Life Technologies, catalog number: 15524-010) 10% Ammonium persulfate (Sigma, catalog number: A3678-100G) (See Recipes)

    Sonication:

    Article Title: The microRNAs in an Ancient Protist Repress the Variant-Specific Surface Protein Expression by Targeting the Entire Coding Sequence
    Article Snippet: For the addition of a sonication step to the procedure, the cells were resuspend in RIPA buffer as described previously and sonicated twice with five 1 second bursts at a 20% duty cycle. .. The purified small RNAs were ligated at the 3′-end to 5–10 µM gel purified miRNA Cloning Linker-1 (IDT) using T4 RNA ligase 2 and truncated with K227Q (NEB) by incubating at 25°C for 1 hr, 15°C for 2 hrs and followed by a final 4°C incubation overnight.

    Affinity Purification:

    Article Title: Transgenerational function of Tetrahymena Piwi protein Twi8p at distinctive noncoding RNA loci
    Article Snippet: Paragraph title: RNP affinity purification, sRNA cloning, and bioinformatic analyses ... Briefly, IDT miRNA cloning linker 1 (Modban) was ligated to the 3′ end of the purified sRNAs using T4 RNA ligase, truncated, K227Q (NEB).

    Recombinant:

    Article Title: Kinetic CRAC uncovers a role for Nab3 in determining gene expression profiles during stress
    Article Snippet: Beads were subsequently incubated with 80 µl of 1xPNK buffer containing eight units of TSAP alkaline phosphatase (Promega) and 80 units of recombinant RNasin (Promega) for 1 h at 37 °C. .. After one 500 µl wash with wash buffer I and three 500 µl washes with 1xPNK buffer, the beads were resuspended in 80 µl of 3′ linker ligation mix (1xPNK buffer, App-PE 3′ adapter (see Supplementary Table ; 0.6 µM final concentration), 10% PEG8000, 30 units of T4 RNA ligase 2 truncated K227Q (NEB), 60 units RNAsin (Promega)).

    Article Title: BioTAP-XL - crosslinking/tandem affinity purification to study DNA targets, RNA and protein components of chromatin associated complexes
    Article Snippet: It allows ligation of Solexa 3′ and 5′ linkers directly to the RNA while still bound to the streptavidin-agarose beads as a part of the crosslinked RNA-protein-DNA complexes recovered from the BioTAP-XL tandem purification. .. CRAC-TN150 buffer (see recipe) CRAC-Wash buffer 1 (see recipe) 1 x CRAC-PNK buffer (see recipe) 5 x CRAC-PNK buffer (see recipe) TruSeq ChIP Sample Prep Kit for ChIP-Seq (Illumina, IP-202-1012) RNace-It Ribonuclease Cocktail 6000U (Agilent Technologies Inc, cat. no. 400720) TSAP Thermosensitive Alkaline Phosphatase (Promega, cat. no. M9910) RNasin Recombinant Ribonuclease Inhibitor (Promega, cat. no. N2515) 10mM dNTP Mix (Invitrogen, cat. no. 18427-088) SuperScript III First-Strand Synthesis kit (Invitrogen, cat. no. 18080-051) Phusion Hot Start II DNA Polymerase 2 U/μL (Thermo Scientific, cat. no. F-549S) T4 RNA ligase 2 truncated K227Q and 50% PEG 8000 (New England BioLabs Inc, cat. no. M0351L) T4 RNA Ligase 1 (New England BioLabs Inc, cat. no. M0204L) T4 PNK-Polynucleotide Kinase from T4-infected Escherichia coli (Sigma, cat. no. P4390-500UN). .. 100 mM ATP (Adenosine 5′-Triphosphate) solution (GE Healthcare Life Sciences, cat. no. 27-2056-01) 3′-BioTAP-Solexa linker-/5rApp/rUrCrGrUrArUrGrCrCrGrUrCrUrUrCrUrGrCrUrUrGrUr/ddC/ This RNA oligo has a blocked 3′ (ddC) end and an ac tivated adenosine at the 5′ end, and may be ordered from BiooScientific.

    ChIP-sequencing:

    Article Title: BioTAP-XL - crosslinking/tandem affinity purification to study DNA targets, RNA and protein components of chromatin associated complexes
    Article Snippet: It allows ligation of Solexa 3′ and 5′ linkers directly to the RNA while still bound to the streptavidin-agarose beads as a part of the crosslinked RNA-protein-DNA complexes recovered from the BioTAP-XL tandem purification. .. CRAC-TN150 buffer (see recipe) CRAC-Wash buffer 1 (see recipe) 1 x CRAC-PNK buffer (see recipe) 5 x CRAC-PNK buffer (see recipe) TruSeq ChIP Sample Prep Kit for ChIP-Seq (Illumina, IP-202-1012) RNace-It Ribonuclease Cocktail 6000U (Agilent Technologies Inc, cat. no. 400720) TSAP Thermosensitive Alkaline Phosphatase (Promega, cat. no. M9910) RNasin Recombinant Ribonuclease Inhibitor (Promega, cat. no. N2515) 10mM dNTP Mix (Invitrogen, cat. no. 18427-088) SuperScript III First-Strand Synthesis kit (Invitrogen, cat. no. 18080-051) Phusion Hot Start II DNA Polymerase 2 U/μL (Thermo Scientific, cat. no. F-549S) T4 RNA ligase 2 truncated K227Q and 50% PEG 8000 (New England BioLabs Inc, cat. no. M0351L) T4 RNA Ligase 1 (New England BioLabs Inc, cat. no. M0204L) T4 PNK-Polynucleotide Kinase from T4-infected Escherichia coli (Sigma, cat. no. P4390-500UN). .. 100 mM ATP (Adenosine 5′-Triphosphate) solution (GE Healthcare Life Sciences, cat. no. 27-2056-01) 3′-BioTAP-Solexa linker-/5rApp/rUrCrGrUrArUrGrCrCrGrUrCrUrUrCrUrGrCrUrUrGrUr/ddC/ This RNA oligo has a blocked 3′ (ddC) end and an ac tivated adenosine at the 5′ end, and may be ordered from BiooScientific.

    RNA Sequencing Assay:

    Article Title: 3′ end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character—RNA-Seq analyses
    Article Snippet: Paragraph title: Library preparation for RNA-Seq ... The 3′ ends of dephosphorylated RNAs were then ligated to 5′ pre-adenylated 3′ adapter with 3′ Biotin modification (the 3′ adapter sequence is shown in ), which was attached to Streptavidin Magnetic Beads (New England BioLabs) , by T4 RNA Ligase 2 truncated K227Q (New England BioLabs) ( ).

    Article Title: Evaluating bias-reducing protocols for RNA sequencing library preparation
    Article Snippet: For standard libraries, ligation of 10 ng of RNA pool was performed with the Ion Total RNA-Seq kit v2 (Life Technologies), following the manufacturer’s instructions. .. Ligation with 200 U T4 RNA Ligase 2 truncated K227Q mutant (trRnl2 K227Q; NEB) was performed in the presence of 1X RNA ligase buffer (NEB), 15% PEG8000, 20 U SuperaseIn (Ambion), at 25°C for 3 hr.

    Article Title: The ribonuclease activity of SAMHD1 is required for HIV-1 restriction
    Article Snippet: Paragraph title: RNA-Seq Analysis ... 3′ adaptor (5′/rApp/TGGAATTCTCGGGTGCCAAGG/ddC/-3′, IDT) was ligated using T4 RNA ligase truncated K227Q (NEB) followed by ligation of 5′ adaptor (5′ Solexa linker, 100-M; 5′rGrUrUrCrArGrArGrUrUrCrUrArCrArGrUrCrCrGrArCrGrA rUrC-3′, IDT) using T4 RNA ligase (TaKaRa).

    Article Title: Nucleotide resolution mapping of influenza A virus nucleoprotein-RNA interactions reveals RNA features required for replication
    Article Snippet: Paragraph title: IAV nucleoprotein PAR-CLIP and RNA-seq library generation ... 3′-OH RNA previously prepared by CIP treatment was ligated to a 3′ adaptor using T4 RNA Ligase 2, truncated K227Q (NEB), according to manufacturer’s instructions.

    Fluorescence:

    Article Title: Histone gene replacement reveals a post-transcriptional role for H3K36 in maintaining metazoan transcriptome fidelity
    Article Snippet: For RT-qPCR, reactions were prepared in biological triplicate using Maxima SYBR Green/ROX qPCR Master Mix (Thermo Scientific), and fluorescence was monitored across 40 cycles in 96 well plate format. .. For LM-PAT, 1 µg total RNA was incubated with 5 pmol preadenylated lmPAT anchor primer (ppApCAGCTGTAGCTATGCGCACCGAGTCAGATCAG) (adenylated using 5’ DNA Adenylation Kit, NEB), and ligated with T4 RNA Ligase 2, truncated K227Q (NEB) using manufacturers protocol.

    Magnetic Beads:

    Article Title: 3′ end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character—RNA-Seq analyses
    Article Snippet: Afterward, phosphatase activity was eliminated by heat inactivation at 65°C for 5 min. .. The 3′ ends of dephosphorylated RNAs were then ligated to 5′ pre-adenylated 3′ adapter with 3′ Biotin modification (the 3′ adapter sequence is shown in ), which was attached to Streptavidin Magnetic Beads (New England BioLabs) , by T4 RNA Ligase 2 truncated K227Q (New England BioLabs) ( ). .. The 40 μl ligation reactions containing 20% (w/v) PEG 8000, 0.05 mg/ml bovine serum albumin, 50 mM NaCl and 2 μl of RNA Ligase 2 at 200 U μl−1 were incubated at 16°C overnight.

    Mutagenesis:

    Article Title: Preparation of Multiplexed Small RNA Libraries From Plants
    Article Snippet: Fiber glass (Corning Incorporated, catalog number: 988-10144) Extra thick blot paper (Bio-Rad Laboratories, catalog number: 170-3969) Whatman DE81 ion exchange cellulose chromatography paper (Thermo Fisher Scientific, catalog number: 05-717-1A) GlycoBlue coprecipitant (Life Technologies, catalog number: AM9516) 100% ethanol 75% ethanol EB buffer (QIAGEN, catalog number: 19086) Qubit RNA HS Assay Kit for quantification of purified small RNAs (Life Technologies, catalog number: ) Qubit dsDNA HS Assay Kit for quantification of DNA amplicon (Life Technologies, catalog number: ) Low salt buffer (see Recipes) High salt buffer (see Recipes) .. 8-Strip PCR thin-walled, 200 μl tubes (Corning Incorporated, Axygen® , catalog number: PCR-0208-CP-C) miRNA cloning linker 1 (/5′App/CTGTAGGCACCATCAAT/3′ddC/; 1 nm) (Integrated DNA Technologies, catalog number: 11-04-03-05) Truncated, K227Q mutation T4 RNA Ligase 2 (New England Biolabs, catalog number: M0351L) De-adenylase (New England Biolabs, catalog number: M0331S) Exonuclease VII (United State Biological, catalog number: 70082Z) RNA 5′ Adapter (GUUCAGAGUUCUACAGUCCGACGAUC) (Illumina RA5, catalog number: 15013205) dATP (Life Technologies, catalog number: 55082) T4 RNA Ligase I (Life Technologies, catalog number: AM2141) RT-PCR Primer (ATTGATGGTGCCTACAG; 25 nmol; de-salted) (Integrated DNA Technologies) SuperScript III (Life Technologies, catalog number: 18080051) Phusion High Fidelity II (Thermo Fisher Scientific, catalog number: F549L) 5′ PCR Primer (Illumina small RNA PCR primer 2: AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA) 3′ Indexed PCR Primer I1 – I12 (100 nmol; PAGE-purified) (Integrated DNA Technologies) Note: The barcodes below (underlined sequences) are reverse complemented in the final Illumina output sequence (see sequence in square brackets for each primer). .. I1 [CGATGT]: CAAGCAGAAGACGGCATACGA ACATCG ATTGATGGTGCCTACAG I2 [GATCAC]: CAAGCAGAAGACGGCATACGA GTGATC ATTGATGGTGCCTACAG I3 [CAGATG]: CAAGCAGAAGACGGCATACGA CATCTG ATTGATGGTGCCTACAG I4 [TACGTT]: CAAGCAGAAGACGGCATACGA AACGTA ATTGATGGTGCCTACAG I5 [TTACCA]: CAAGCAGAAGACGGCATACGA TGGTAA ATTGATGGTGCCTACAG I6 [ACTGTA]: CAAGCAGAAGACGGCATACGA TACAGT ATTGATGGTGCCTACAG I7 [ATCACG]: CAAGCAGAAGACGGCATACGA CGTGAT ATTGATGGTGCCTACAG I8 [ACTTGT]: CAAGCAGAAGACGGCATACGA ACAAGT ATTGATGGTGCCTACAG I9 [GCCAAT]: CAAGCAGAAGACGGCATACGA ATTGGC ATTGATGGTGCCTACAG I10 [TGCTAG]: CAAGCAGAAGACGGCATACGA CTAGCA ATTGATGGTGCCTACAG I11 [CTTGTA]: CAAGCAGAAGACGGCATACGA TACAAG ATTGATGGTGCCTACAG I12 [TCAGGC]: CAAGCAGAAGACGGCATACGA GCCTGA ATTGATGGTGCCTACAG QIAquick PCR Purification Kit (QIAGEN, catalog number: 28106) Diethylpyrocarbonate (DEPC)-H2 O/ RNase-free H2 O DNA size marker suitable for detecting a ~120 bp fragment (50 bp step ladder, Promega Corporation, catalog number: G4521; 25 bp step ladder, Promega Corporation, catalog number: G4511) 3 M sodium acetate (NaOAc) (pH 5.5) Reagents for 6% native PAGE 37.5: 1 polyacrylamide:bisacrylamide (see Recipes) TEMED (Life Technologies, catalog number: 15524-010) 10% Ammonium persulfate (Sigma, catalog number: A3678-100G) (See Recipes)

    Article Title: The Coding Regions of Germline mRNAs Confer Sensitivity to Argonaute Regulation in C. elegans
    Article Snippet: Small RNAs were further enriched using MirVana Kit (Thermo Fisher Scientific). .. Samples from wild-type and mutant were pretreated with a homemade 5′ polyphosphatase and were ligated to the 3′ adaptor linker 1 (5′ rAppAGATCGGAAGAGCACACGTCTGAACTCCAGTCA/3ddC/3′, IDT) using T4 RNA ligase 2 (M0351S, NEB). .. Subsequently, the 5′ adaptor (rArCrA rCrUrCrUrUrUrCrCrCrUrArCrArCrGrArCrGrCrUrCrUrUrCrCrGrArUrCrU) was ligated using T4 RNA ligase 1 (M0204S, NEB).

    Article Title: Evaluating bias-reducing protocols for RNA sequencing library preparation
    Article Snippet: For the CircLig libraries, 40 ng RNA pool (~6 pmol) and 100 ng Universal Cloning Linker (~18 pmol; NEB) were denatured at 80°C for 2 min then placed immediately on ice. .. Ligation with 200 U T4 RNA Ligase 2 truncated K227Q mutant (trRnl2 K227Q; NEB) was performed in the presence of 1X RNA ligase buffer (NEB), 15% PEG8000, 20 U SuperaseIn (Ambion), at 25°C for 3 hr. .. Ligation with 20 pmol Mth K97A was performed in 1X NEB1 buffer and 20 U SuperaseIn, at 65°C for 1 hr, as described by Zhelkovsky and McReynolds (11).

    Isolation:

    Article Title: Biased transcription and selective degradation of small RNAs shape the pattern of DNA elimination in Tetrahymena
    Article Snippet: After DNase I treatment, the total RNA was isolated with TRIzol LS reagent (Invitrogen) and resuspended in 20 μL of water. .. For 3′ adapter ligation, 10 μL of the end-repaired RNAs was incubated with 1 μL of 50 μM 3′ adapter, 0.5 μL of Ribolock (Fermentas), 2.15 μL of ATP-free T4 RNA ligase buffer, 6 μL of 50% PEG-8000, and 2 μL of T4 RNA ligase 2-truncated K227Q (New England Biolabs) overnight at 16°C.

    Article Title: The Gcn4 transcription factor reduces protein synthesis capacity and extends yeast lifespan
    Article Snippet: In brief, after isolation, 50 ng of mRNA was chemically fragmented by incubating the mRNA solution with twice the volume of alkaline hydrolysis buffer (50 mM sodium carbonate [NaHCO3 /Na2 CO3 ] pH 9.2, 1 mM EDTA) at 95 °C for 5 min to obtain fragments of ~200–300 bases. .. Ligation of RNA 3′ adapter (RA3, part # 15013207, Illumina) was done using T4 RNA Ligase 2, truncated K227Q (M0351L, New England Biolabs Inc) according to the Illumina protocol.

    Article Title: Global analysis of CPSF2-mediated alternative splicing: Integration of global iCLIP and transcriptome profiling data
    Article Snippet: To ligate linkers to the 3′ end of RNAs, 4.5 μl of purified RNA was mixed with 1 μl of 10 μM miRCat33 Adaptor (Integrated DNA Technologies), 1.5 μl of RNA Ligase buffer, 0.5 μl of T4 RNA Ligase 2, Truncated K227Q (NEB), and 7.5 μl of PEG 8000, and incubated at 30 °C for 6 h. Samples were then end-labeled using PNK enzyme and run on a NuPAGE Novex 10% Bis–Tris Protein Gel (Invitrogen) with 1 × MOPS running buffer (Invitrogen). .. To ligate linkers to the 3′ end of RNAs, 4.5 μl of purified RNA was mixed with 1 μl of 10 μM miRCat33 Adaptor (Integrated DNA Technologies), 1.5 μl of RNA Ligase buffer, 0.5 μl of T4 RNA Ligase 2, Truncated K227Q (NEB), and 7.5 μl of PEG 8000, and incubated at 30 °C for 6 h. Samples were then end-labeled using PNK enzyme and run on a NuPAGE Novex 10% Bis–Tris Protein Gel (Invitrogen) with 1 × MOPS running buffer (Invitrogen).

    Article Title: Nucleotide resolution mapping of influenza A virus nucleoprotein-RNA interactions reveals RNA features required for replication
    Article Snippet: To account for potential bias introduced during adaptor ligation and sequencing, we also generated RNA-seq libraries using RNA extracted from uncross-linked cell lysates of influenza infected 293T cells from which RNA was isolated by TRIzol (Invitrogen) extraction, then 10 μg RNA was fragmented by Mg2+ at 95 °C for 12 min (NEB, Magnesium RNA Fragmentation Module). .. 3′-OH RNA previously prepared by CIP treatment was ligated to a 3′ adaptor using T4 RNA Ligase 2, truncated K227Q (NEB), according to manufacturer’s instructions.

    Article Title: The microRNAs in an Ancient Protist Repress the Variant-Specific Surface Protein Expression by Targeting the Entire Coding Sequence
    Article Snippet: Briefly, the GlAgo associated small RNAs were isolated from Giardia WB trophozoites as previously described and resuspended in 10 µl RNase-free diH2 O . .. The purified small RNAs were ligated at the 3′-end to 5–10 µM gel purified miRNA Cloning Linker-1 (IDT) using T4 RNA ligase 2 and truncated with K227Q (NEB) by incubating at 25°C for 1 hr, 15°C for 2 hrs and followed by a final 4°C incubation overnight.

    Purification:

    Article Title: Biased transcription and selective degradation of small RNAs shape the pattern of DNA elimination in Tetrahymena
    Article Snippet: The RNAs were treated with DNase I again, and RNA was purified twice through P30 spin columns (Bio-Rad). .. For 3′ adapter ligation, 10 μL of the end-repaired RNAs was incubated with 1 μL of 50 μM 3′ adapter, 0.5 μL of Ribolock (Fermentas), 2.15 μL of ATP-free T4 RNA ligase buffer, 6 μL of 50% PEG-8000, and 2 μL of T4 RNA ligase 2-truncated K227Q (New England Biolabs) overnight at 16°C.

    Article Title: The Gcn4 transcription factor reduces protein synthesis capacity and extends yeast lifespan
    Article Snippet: The fragmented mRNA was further incubated with ATP and T4 polynucleotide kinase (EK0032, Fermentas) at 37 °C for 1 h and subsequently purified. .. Ligation of RNA 3′ adapter (RA3, part # 15013207, Illumina) was done using T4 RNA Ligase 2, truncated K227Q (M0351L, New England Biolabs Inc) according to the Illumina protocol.

    Article Title: Global analysis of CPSF2-mediated alternative splicing: Integration of global iCLIP and transcriptome profiling data
    Article Snippet: Samples were then washed twice with high-salt wash buffer and once with 900 μl PNK wash buffer. .. To ligate linkers to the 3′ end of RNAs, 4.5 μl of purified RNA was mixed with 1 μl of 10 μM miRCat33 Adaptor (Integrated DNA Technologies), 1.5 μl of RNA Ligase buffer, 0.5 μl of T4 RNA Ligase 2, Truncated K227Q (NEB), and 7.5 μl of PEG 8000, and incubated at 30 °C for 6 h. Samples were then end-labeled using PNK enzyme and run on a NuPAGE Novex 10% Bis–Tris Protein Gel (Invitrogen) with 1 × MOPS running buffer (Invitrogen). .. Proteins and covalently bound RNAs were then transferred to a nitrocellulose membrane (Whatman) using a Novex wet transfer apparatus (Invitrogen).

    Article Title: Single‐cell mRNA profiling reveals the hierarchical response of mi RNA targets to mi RNA induction
    Article Snippet: Purified mRNA fragments were dephosphorylated with FastAP (Life Technologies, Inc.) and 5′‐phosphorylated with PNK (Life Technologies, Inc.) following manufacturer's instructions for optimal conditions of the enzymatic reaction. .. After another round of RNeasy MinElute Cleanup Kit (Qiagen, Inc.), a pre‐adenylated DNA adapter (5′‐TGGAATTCTCGGGTGCCAAGG‐3′) was ligated to the 3′ end of the mRNA fragments at 4°C overnight using the T4 RNA ligase 2, truncated K227Q (New England Biolabs, Inc.), in 1× T4 RNA ligase buffer (no ATP) and 15% DMSO.

    Article Title: The microRNAs in an Ancient Protist Repress the Variant-Specific Surface Protein Expression by Targeting the Entire Coding Sequence
    Article Snippet: For the addition of a sonication step to the procedure, the cells were resuspend in RIPA buffer as described previously and sonicated twice with five 1 second bursts at a 20% duty cycle. .. The purified small RNAs were ligated at the 3′-end to 5–10 µM gel purified miRNA Cloning Linker-1 (IDT) using T4 RNA ligase 2 and truncated with K227Q (NEB) by incubating at 25°C for 1 hr, 15°C for 2 hrs and followed by a final 4°C incubation overnight. .. The reaction mixture was analyzed in 15% polyacrylamide/8 M Urea gel, and the band of ligated product was excised.

    Article Title: Transgenerational function of Tetrahymena Piwi protein Twi8p at distinctive noncoding RNA loci
    Article Snippet: Size selection of purified sRNAs was performed by denaturing PAGE. sRNAs were prepared for Solexa sequencing as previously described ( ). .. Briefly, IDT miRNA cloning linker 1 (Modban) was ligated to the 3′ end of the purified sRNAs using T4 RNA ligase, truncated, K227Q (NEB). .. Ligations were incubated at 16°C overnight to minimize the impact of 3′-terminal 2′ O -methylation on the sRNAs ( ).

    Sequencing:

    Article Title: Biased transcription and selective degradation of small RNAs shape the pattern of DNA elimination in Tetrahymena
    Article Snippet: A 20-μL aliquot of the RNA was end-repaired and resuspended in 10.5 μL of water. cDNA was synthesized as described above in the section “Small RNA cDNA Library Preparation and Sequencing” with some modifications. .. For 3′ adapter ligation, 10 μL of the end-repaired RNAs was incubated with 1 μL of 50 μM 3′ adapter, 0.5 μL of Ribolock (Fermentas), 2.15 μL of ATP-free T4 RNA ligase buffer, 6 μL of 50% PEG-8000, and 2 μL of T4 RNA ligase 2-truncated K227Q (New England Biolabs) overnight at 16°C.

    Article Title: 3′ end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character—RNA-Seq analyses
    Article Snippet: Afterward, phosphatase activity was eliminated by heat inactivation at 65°C for 5 min. .. The 3′ ends of dephosphorylated RNAs were then ligated to 5′ pre-adenylated 3′ adapter with 3′ Biotin modification (the 3′ adapter sequence is shown in ), which was attached to Streptavidin Magnetic Beads (New England BioLabs) , by T4 RNA Ligase 2 truncated K227Q (New England BioLabs) ( ). .. The 40 μl ligation reactions containing 20% (w/v) PEG 8000, 0.05 mg/ml bovine serum albumin, 50 mM NaCl and 2 μl of RNA Ligase 2 at 200 U μl−1 were incubated at 16°C overnight.

    Article Title: The Gcn4 transcription factor reduces protein synthesis capacity and extends yeast lifespan
    Article Snippet: Paragraph title: mRNA sequencing ... Ligation of RNA 3′ adapter (RA3, part # 15013207, Illumina) was done using T4 RNA Ligase 2, truncated K227Q (M0351L, New England Biolabs Inc) according to the Illumina protocol.

    Article Title: Preparation of Multiplexed Small RNA Libraries From Plants
    Article Snippet: Fiber glass (Corning Incorporated, catalog number: 988-10144) Extra thick blot paper (Bio-Rad Laboratories, catalog number: 170-3969) Whatman DE81 ion exchange cellulose chromatography paper (Thermo Fisher Scientific, catalog number: 05-717-1A) GlycoBlue coprecipitant (Life Technologies, catalog number: AM9516) 100% ethanol 75% ethanol EB buffer (QIAGEN, catalog number: 19086) Qubit RNA HS Assay Kit for quantification of purified small RNAs (Life Technologies, catalog number: ) Qubit dsDNA HS Assay Kit for quantification of DNA amplicon (Life Technologies, catalog number: ) Low salt buffer (see Recipes) High salt buffer (see Recipes) .. 8-Strip PCR thin-walled, 200 μl tubes (Corning Incorporated, Axygen® , catalog number: PCR-0208-CP-C) miRNA cloning linker 1 (/5′App/CTGTAGGCACCATCAAT/3′ddC/; 1 nm) (Integrated DNA Technologies, catalog number: 11-04-03-05) Truncated, K227Q mutation T4 RNA Ligase 2 (New England Biolabs, catalog number: M0351L) De-adenylase (New England Biolabs, catalog number: M0331S) Exonuclease VII (United State Biological, catalog number: 70082Z) RNA 5′ Adapter (GUUCAGAGUUCUACAGUCCGACGAUC) (Illumina RA5, catalog number: 15013205) dATP (Life Technologies, catalog number: 55082) T4 RNA Ligase I (Life Technologies, catalog number: AM2141) RT-PCR Primer (ATTGATGGTGCCTACAG; 25 nmol; de-salted) (Integrated DNA Technologies) SuperScript III (Life Technologies, catalog number: 18080051) Phusion High Fidelity II (Thermo Fisher Scientific, catalog number: F549L) 5′ PCR Primer (Illumina small RNA PCR primer 2: AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA) 3′ Indexed PCR Primer I1 – I12 (100 nmol; PAGE-purified) (Integrated DNA Technologies) Note: The barcodes below (underlined sequences) are reverse complemented in the final Illumina output sequence (see sequence in square brackets for each primer). .. I1 [CGATGT]: CAAGCAGAAGACGGCATACGA ACATCG ATTGATGGTGCCTACAG I2 [GATCAC]: CAAGCAGAAGACGGCATACGA GTGATC ATTGATGGTGCCTACAG I3 [CAGATG]: CAAGCAGAAGACGGCATACGA CATCTG ATTGATGGTGCCTACAG I4 [TACGTT]: CAAGCAGAAGACGGCATACGA AACGTA ATTGATGGTGCCTACAG I5 [TTACCA]: CAAGCAGAAGACGGCATACGA TGGTAA ATTGATGGTGCCTACAG I6 [ACTGTA]: CAAGCAGAAGACGGCATACGA TACAGT ATTGATGGTGCCTACAG I7 [ATCACG]: CAAGCAGAAGACGGCATACGA CGTGAT ATTGATGGTGCCTACAG I8 [ACTTGT]: CAAGCAGAAGACGGCATACGA ACAAGT ATTGATGGTGCCTACAG I9 [GCCAAT]: CAAGCAGAAGACGGCATACGA ATTGGC ATTGATGGTGCCTACAG I10 [TGCTAG]: CAAGCAGAAGACGGCATACGA CTAGCA ATTGATGGTGCCTACAG I11 [CTTGTA]: CAAGCAGAAGACGGCATACGA TACAAG ATTGATGGTGCCTACAG I12 [TCAGGC]: CAAGCAGAAGACGGCATACGA GCCTGA ATTGATGGTGCCTACAG QIAquick PCR Purification Kit (QIAGEN, catalog number: 28106) Diethylpyrocarbonate (DEPC)-H2 O/ RNase-free H2 O DNA size marker suitable for detecting a ~120 bp fragment (50 bp step ladder, Promega Corporation, catalog number: G4521; 25 bp step ladder, Promega Corporation, catalog number: G4511) 3 M sodium acetate (NaOAc) (pH 5.5) Reagents for 6% native PAGE 37.5: 1 polyacrylamide:bisacrylamide (see Recipes) TEMED (Life Technologies, catalog number: 15524-010) 10% Ammonium persulfate (Sigma, catalog number: A3678-100G) (See Recipes)

    Article Title: The Coding Regions of Germline mRNAs Confer Sensitivity to Argonaute Regulation in C. elegans
    Article Snippet: Paragraph title: Small RNA Cloning and Deep Sequencing ... Samples from wild-type and mutant were pretreated with a homemade 5′ polyphosphatase and were ligated to the 3′ adaptor linker 1 (5′ rAppAGATCGGAAGAGCACACGTCTGAACTCCAGTCA/3ddC/3′, IDT) using T4 RNA ligase 2 (M0351S, NEB).

    Article Title: Oligoadenylation of 3′ decay intermediates promotes cytoplasmic mRNA degradation in Drosophila cells
    Article Snippet: Paragraph title: Illumina sequencing ... In the second data set, truncated T4 RNA Ligase 2 K227Q (NEB) was used for the same type of control.

    Article Title: The ribonuclease activity of SAMHD1 is required for HIV-1 restriction
    Article Snippet: 3′ adaptor (5′/rApp/TGGAATTCTCGGGTGCCAAGG/ddC/-3′, IDT) was ligated using T4 RNA ligase truncated K227Q (NEB) followed by ligation of 5′ adaptor (5′ Solexa linker, 100-M; 5′rGrUrUrCrArGrArGrUrUrCrUrArCrArGrUrCrCrGrArCrGrA rUrC-3′, IDT) using T4 RNA ligase (TaKaRa). .. 3′ adaptor (5′/rApp/TGGAATTCTCGGGTGCCAAGG/ddC/-3′, IDT) was ligated using T4 RNA ligase truncated K227Q (NEB) followed by ligation of 5′ adaptor (5′ Solexa linker, 100-M; 5′rGrUrUrCrArGrArGrUrUrCrUrArCrArGrUrCrCrGrArCrGrA rUrC-3′, IDT) using T4 RNA ligase (TaKaRa).

    Article Title: Nucleotide resolution mapping of influenza A virus nucleoprotein-RNA interactions reveals RNA features required for replication
    Article Snippet: In both cases, total RNA was precipitated with ethanol, and used to prepare Illumina sequencing libraries. .. 3′-OH RNA previously prepared by CIP treatment was ligated to a 3′ adaptor using T4 RNA Ligase 2, truncated K227Q (NEB), according to manufacturer’s instructions.

    Article Title: Transgenerational function of Tetrahymena Piwi protein Twi8p at distinctive noncoding RNA loci
    Article Snippet: Size selection of purified sRNAs was performed by denaturing PAGE. sRNAs were prepared for Solexa sequencing as previously described ( ). .. Briefly, IDT miRNA cloning linker 1 (Modban) was ligated to the 3′ end of the purified sRNAs using T4 RNA ligase, truncated, K227Q (NEB).

    Quantitative RT-PCR:

    Article Title: Histone gene replacement reveals a post-transcriptional role for H3K36 in maintaining metazoan transcriptome fidelity
    Article Snippet: For RT-qPCR, reactions were prepared in biological triplicate using Maxima SYBR Green/ROX qPCR Master Mix (Thermo Scientific), and fluorescence was monitored across 40 cycles in 96 well plate format. .. For LM-PAT, 1 µg total RNA was incubated with 5 pmol preadenylated lmPAT anchor primer (ppApCAGCTGTAGCTATGCGCACCGAGTCAGATCAG) (adenylated using 5’ DNA Adenylation Kit, NEB), and ligated with T4 RNA Ligase 2, truncated K227Q (NEB) using manufacturers protocol.

    De-Phosphorylation Assay:

    Article Title: The ribonuclease activity of SAMHD1 is required for HIV-1 restriction
    Article Snippet: After dephosphorylation by Antarctic phosphatase (NEB), 5′ end of fragmented and dephosphorylated RNA was phospho-labeled using [γ-32 P]ATP and T4 polynucleotide kinase (TaKaRa). .. 3′ adaptor (5′/rApp/TGGAATTCTCGGGTGCCAAGG/ddC/-3′, IDT) was ligated using T4 RNA ligase truncated K227Q (NEB) followed by ligation of 5′ adaptor (5′ Solexa linker, 100-M; 5′rGrUrUrCrArGrArGrUrUrCrUrArCrArGrUrCrCrGrArCrGrA rUrC-3′, IDT) using T4 RNA ligase (TaKaRa).

    Nested PCR:

    Article Title: Oligoadenylation of 3′ decay intermediates promotes cytoplasmic mRNA degradation in Drosophila cells
    Article Snippet: In the second data set, truncated T4 RNA Ligase 2 K227Q (NEB) was used for the same type of control. .. After G50 gel filtration, phenol–chloroform extraction and ethanol precipitation, ligation products were reverse-transcribed (M-MLV reverse transcriptase, Promega) with an adaptor reverse oligonucleotide.

    Sample Prep:

    Article Title: The Gcn4 transcription factor reduces protein synthesis capacity and extends yeast lifespan
    Article Snippet: Libraries for mRNA sequencing were prepared using the “directional mRNA-seq sample preparation” protocol from Illumina, with minor modifications. .. Ligation of RNA 3′ adapter (RA3, part # 15013207, Illumina) was done using T4 RNA Ligase 2, truncated K227Q (M0351L, New England Biolabs Inc) according to the Illumina protocol.

    Article Title: BioTAP-XL - crosslinking/tandem affinity purification to study DNA targets, RNA and protein components of chromatin associated complexes
    Article Snippet: It allows ligation of Solexa 3′ and 5′ linkers directly to the RNA while still bound to the streptavidin-agarose beads as a part of the crosslinked RNA-protein-DNA complexes recovered from the BioTAP-XL tandem purification. .. CRAC-TN150 buffer (see recipe) CRAC-Wash buffer 1 (see recipe) 1 x CRAC-PNK buffer (see recipe) 5 x CRAC-PNK buffer (see recipe) TruSeq ChIP Sample Prep Kit for ChIP-Seq (Illumina, IP-202-1012) RNace-It Ribonuclease Cocktail 6000U (Agilent Technologies Inc, cat. no. 400720) TSAP Thermosensitive Alkaline Phosphatase (Promega, cat. no. M9910) RNasin Recombinant Ribonuclease Inhibitor (Promega, cat. no. N2515) 10mM dNTP Mix (Invitrogen, cat. no. 18427-088) SuperScript III First-Strand Synthesis kit (Invitrogen, cat. no. 18080-051) Phusion Hot Start II DNA Polymerase 2 U/μL (Thermo Scientific, cat. no. F-549S) T4 RNA ligase 2 truncated K227Q and 50% PEG 8000 (New England BioLabs Inc, cat. no. M0351L) T4 RNA Ligase 1 (New England BioLabs Inc, cat. no. M0204L) T4 PNK-Polynucleotide Kinase from T4-infected Escherichia coli (Sigma, cat. no. P4390-500UN). .. 100 mM ATP (Adenosine 5′-Triphosphate) solution (GE Healthcare Life Sciences, cat. no. 27-2056-01) 3′-BioTAP-Solexa linker-/5rApp/rUrCrGrUrArUrGrCrCrGrUrCrUrUrCrUrGrCrUrUrGrUr/ddC/ This RNA oligo has a blocked 3′ (ddC) end and an ac tivated adenosine at the 5′ end, and may be ordered from BiooScientific.

    Chromatin Immunoprecipitation:

    Article Title: BioTAP-XL - crosslinking/tandem affinity purification to study DNA targets, RNA and protein components of chromatin associated complexes
    Article Snippet: It allows ligation of Solexa 3′ and 5′ linkers directly to the RNA while still bound to the streptavidin-agarose beads as a part of the crosslinked RNA-protein-DNA complexes recovered from the BioTAP-XL tandem purification. .. CRAC-TN150 buffer (see recipe) CRAC-Wash buffer 1 (see recipe) 1 x CRAC-PNK buffer (see recipe) 5 x CRAC-PNK buffer (see recipe) TruSeq ChIP Sample Prep Kit for ChIP-Seq (Illumina, IP-202-1012) RNace-It Ribonuclease Cocktail 6000U (Agilent Technologies Inc, cat. no. 400720) TSAP Thermosensitive Alkaline Phosphatase (Promega, cat. no. M9910) RNasin Recombinant Ribonuclease Inhibitor (Promega, cat. no. N2515) 10mM dNTP Mix (Invitrogen, cat. no. 18427-088) SuperScript III First-Strand Synthesis kit (Invitrogen, cat. no. 18080-051) Phusion Hot Start II DNA Polymerase 2 U/μL (Thermo Scientific, cat. no. F-549S) T4 RNA ligase 2 truncated K227Q and 50% PEG 8000 (New England BioLabs Inc, cat. no. M0351L) T4 RNA Ligase 1 (New England BioLabs Inc, cat. no. M0204L) T4 PNK-Polynucleotide Kinase from T4-infected Escherichia coli (Sigma, cat. no. P4390-500UN). .. 100 mM ATP (Adenosine 5′-Triphosphate) solution (GE Healthcare Life Sciences, cat. no. 27-2056-01) 3′-BioTAP-Solexa linker-/5rApp/rUrCrGrUrArUrGrCrCrGrUrCrUrUrCrUrGrCrUrUrGrUr/ddC/ This RNA oligo has a blocked 3′ (ddC) end and an ac tivated adenosine at the 5′ end, and may be ordered from BiooScientific.

    SDS Page:

    Article Title: Nucleotide resolution mapping of influenza A virus nucleoprotein-RNA interactions reveals RNA features required for replication
    Article Snippet: Briefly, protein–RNA complexes were separated on 4–12% SDS-PAGE gels and transferred to nitrocellulose membranes. .. 3′-OH RNA previously prepared by CIP treatment was ligated to a 3′ adaptor using T4 RNA Ligase 2, truncated K227Q (NEB), according to manufacturer’s instructions.

    Software:

    Article Title: The ribonuclease activity of SAMHD1 is required for HIV-1 restriction
    Article Snippet: 3′ adaptor (5′/rApp/TGGAATTCTCGGGTGCCAAGG/ddC/-3′, IDT) was ligated using T4 RNA ligase truncated K227Q (NEB) followed by ligation of 5′ adaptor (5′ Solexa linker, 100-M; 5′rGrUrUrCrArGrArGrUrUrCrUrArCrArGrUrCrCrGrArCrGrA rUrC-3′, IDT) using T4 RNA ligase (TaKaRa). .. 3′ adaptor (5′/rApp/TGGAATTCTCGGGTGCCAAGG/ddC/-3′, IDT) was ligated using T4 RNA ligase truncated K227Q (NEB) followed by ligation of 5′ adaptor (5′ Solexa linker, 100-M; 5′rGrUrUrCrArGrArGrUrUrCrUrArCrArGrUrCrCrGrArCrGrA rUrC-3′, IDT) using T4 RNA ligase (TaKaRa).

    Multiplex Assay:

    Article Title: Transgenerational function of Tetrahymena Piwi protein Twi8p at distinctive noncoding RNA loci
    Article Snippet: Briefly, IDT miRNA cloning linker 1 (Modban) was ligated to the 3′ end of the purified sRNAs using T4 RNA ligase, truncated, K227Q (NEB). .. The sequencing adaptor was ligated to sRNA 5′ ends by T4 RNA ligase 1, and the intended products were gel-purified and collected by ethanol precipitation.

    Selection:

    Article Title: Transgenerational function of Tetrahymena Piwi protein Twi8p at distinctive noncoding RNA loci
    Article Snippet: Size selection of purified sRNAs was performed by denaturing PAGE. sRNAs were prepared for Solexa sequencing as previously described ( ). .. Briefly, IDT miRNA cloning linker 1 (Modban) was ligated to the 3′ end of the purified sRNAs using T4 RNA ligase, truncated, K227Q (NEB).

    Agarose Gel Electrophoresis:

    Article Title: Histone gene replacement reveals a post-transcriptional role for H3K36 in maintaining metazoan transcriptome fidelity
    Article Snippet: Reactions were run on a 2% agarose gel with EtBr for 30 min at 90 V, and bands were imaged on a UV transilluminator (GE Healthcare) and quantified using ImageJ. .. For LM-PAT, 1 µg total RNA was incubated with 5 pmol preadenylated lmPAT anchor primer (ppApCAGCTGTAGCTATGCGCACCGAGTCAGATCAG) (adenylated using 5’ DNA Adenylation Kit, NEB), and ligated with T4 RNA Ligase 2, truncated K227Q (NEB) using manufacturers protocol.

    Ethanol Precipitation:

    Article Title: The use of duplex-specific nuclease in ribosome profiling and a user-friendly software package for Ribo-seq data analysis
    Article Snippet: The RNA samples from above were heated at 80°C for 2 min, cooled and the 3′ phosphate group removed using T4 polynucleotide kinase (T4 PNK, New England BioLabs) for 2 h at 37°C in a 20 µL reaction lacking ATP. .. The RNA was concentrated by ethanol precipitation, resuspended in 10 mM Tris–HCl pH 7.5 and ligated in a 20 µL reaction overnight at 14°C to a preadenylated 3′-adaptor (5′-rATGGAATTCTCGGGTGCCAAGG-3′) using T4 RNA Ligase 2 truncated K227Q (New England BioLabs). .. This 3′ adaptor was adenylated using a 5′ DNA adenylation kit (New England BioLabs) following the manufacturer's instructions.

    Article Title: A Novel Epigenetic Silencing Pathway Involving the Highly Conserved 5’-3’ Exoribonuclease Dhp1/Rat1/Xrn2 in Schizosaccharomyces pombe
    Article Snippet: All washes, alkaline phosphatase treatment and 3’ linker ligation were carried out as described except that 40U T4 RNA ligase 2 truncated K227Q (NEB) was used instead of T4 RNA ligase. .. All washes, alkaline phosphatase treatment and 3’ linker ligation were carried out as described except that 40U T4 RNA ligase 2 truncated K227Q (NEB) was used instead of T4 RNA ligase.

    Article Title: Transgenerational function of Tetrahymena Piwi protein Twi8p at distinctive noncoding RNA loci
    Article Snippet: Purified sRNAs were collected by phenol–choloroform extraction followed by ethanol precipitation. .. Briefly, IDT miRNA cloning linker 1 (Modban) was ligated to the 3′ end of the purified sRNAs using T4 RNA ligase, truncated, K227Q (NEB).

    Next-Generation Sequencing:

    Article Title: The microRNAs in an Ancient Protist Repress the Variant-Specific Surface Protein Expression by Targeting the Entire Coding Sequence
    Article Snippet: Paragraph title: NGS library construction ... The purified small RNAs were ligated at the 3′-end to 5–10 µM gel purified miRNA Cloning Linker-1 (IDT) using T4 RNA ligase 2 and truncated with K227Q (NEB) by incubating at 25°C for 1 hr, 15°C for 2 hrs and followed by a final 4°C incubation overnight.

    Immunoprecipitation:

    Article Title: Global analysis of CPSF2-mediated alternative splicing: Integration of global iCLIP and transcriptome profiling data
    Article Snippet: Paragraph title: Individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) ... To ligate linkers to the 3′ end of RNAs, 4.5 μl of purified RNA was mixed with 1 μl of 10 μM miRCat33 Adaptor (Integrated DNA Technologies), 1.5 μl of RNA Ligase buffer, 0.5 μl of T4 RNA Ligase 2, Truncated K227Q (NEB), and 7.5 μl of PEG 8000, and incubated at 30 °C for 6 h. Samples were then end-labeled using PNK enzyme and run on a NuPAGE Novex 10% Bis–Tris Protein Gel (Invitrogen) with 1 × MOPS running buffer (Invitrogen).

    Construct:

    Article Title: The microRNAs in an Ancient Protist Repress the Variant-Specific Surface Protein Expression by Targeting the Entire Coding Sequence
    Article Snippet: The NGS libraries of GlAgo associated small RNAs were constructed using a modified version of the Mello Lab protocol ( www.lsi.umich.edu/files/SmallRNACloning.pdf ). .. The purified small RNAs were ligated at the 3′-end to 5–10 µM gel purified miRNA Cloning Linker-1 (IDT) using T4 RNA ligase 2 and truncated with K227Q (NEB) by incubating at 25°C for 1 hr, 15°C for 2 hrs and followed by a final 4°C incubation overnight.

    Marker:

    Article Title: Evaluating bias-reducing protocols for RNA sequencing library preparation
    Article Snippet: Ligation with 200 U T4 RNA Ligase 2 truncated K227Q mutant (trRnl2 K227Q; NEB) was performed in the presence of 1X RNA ligase buffer (NEB), 15% PEG8000, 20 U SuperaseIn (Ambion), at 25°C for 3 hr. .. Ligation with 200 U T4 RNA Ligase 2 truncated K227Q mutant (trRnl2 K227Q; NEB) was performed in the presence of 1X RNA ligase buffer (NEB), 15% PEG8000, 20 U SuperaseIn (Ambion), at 25°C for 3 hr.

    Lysis:

    Article Title: A Novel Epigenetic Silencing Pathway Involving the Highly Conserved 5’-3’ Exoribonuclease Dhp1/Rat1/Xrn2 in Schizosaccharomyces pombe
    Article Snippet: After two washes with TN1000 buffer (100 mM Tris-HCl (pH 7.8), 2 M NaCl, 0.2% NP-40) and two washes with TN150 lysis buffer, the beads were incubated with GST-TEV protease for 2h at 16°C. .. All washes, alkaline phosphatase treatment and 3’ linker ligation were carried out as described except that 40U T4 RNA ligase 2 truncated K227Q (NEB) was used instead of T4 RNA ligase.

    Article Title: Global analysis of CPSF2-mediated alternative splicing: Integration of global iCLIP and transcriptome profiling data
    Article Snippet: The beads were rotated at room temperature for 60 min, washed twice with lysis buffer, and added to the cross-linked lysate. .. To ligate linkers to the 3′ end of RNAs, 4.5 μl of purified RNA was mixed with 1 μl of 10 μM miRCat33 Adaptor (Integrated DNA Technologies), 1.5 μl of RNA Ligase buffer, 0.5 μl of T4 RNA Ligase 2, Truncated K227Q (NEB), and 7.5 μl of PEG 8000, and incubated at 30 °C for 6 h. Samples were then end-labeled using PNK enzyme and run on a NuPAGE Novex 10% Bis–Tris Protein Gel (Invitrogen) with 1 × MOPS running buffer (Invitrogen).

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    New England Biolabs peg 8000
    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 ) <t>PEG</t> 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.
    Peg 8000, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 75/100, based on 0 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs sterile filtered peg mw 8000
    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 ) <t>PEG</t> 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.
    Sterile Filtered Peg Mw 8000, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sterile filtered peg mw 8000/product/New England Biolabs
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    83
    New England Biolabs peg mw 8000
    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 ) <t>PEG</t> 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.
    Peg Mw 8000, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 83/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/peg mw 8000/product/New England Biolabs
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    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: One milliliter of 5X TEDA solution contained 0.5 M Tris–HCl pH 7.5, 50 mM MgCl2 , 50 mM dithiothreitol, 0.25 g of PEG 8000, and 1 μl of 10 U/μl T5 exonuclease (New England Biolab, Beijing).

    Techniques: Clone Assay