Recjf

Manufactured by New England Biolabs

Sourced in United States

RecJf is a proprietary DNA recombinase enzyme from New England Biolabs. It catalyzes the joining of DNA fragments in a rapid, efficient, and seamless manner.

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RecJf exonuclease (4 citations)

17 protocols using recjf

Quantification of HBV cccDNA and Total DNA

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Total HBV DNA and cccDNA per cell were quantified by cinqPCR (which is compatible with the Genotype D HBV strain used in this project), as previously described.⁶ (link) Total HBV DNA extracts (1 μg) were digested with HhaI (New England Biolabs [NEB]) which excises a fragment proximal to the DNA nick region present in HBV rcDNA (nt1801-nt2800). The 5ʹ→3ʹ single-stranded DNA exonuclease Recj_f (NEB) was added to digest the single-stranded region of rcDNA, which is exposed by unstable binding of the DNA fragment between the DNA nick and the HhaI cut site.
cccDNA (lacking a DNA nick) remains stable and is impervious to Recj_f activity. T4 DNA ligase (NEB) was used to induce intra-molecular ligation of cccDNA fragments (which retain intact complimentary sticky ends), but not rcDNA fragments (which have 1 sticky end digested away by Recj_f). The DNA rings were then linearised by digestion with XbaI (NEB) and then amplified using digital droplet PCR with specific primers to the cccDNA fragment and RNaseP (a single-copy cellular gene). Throughout this procedure, dslDNA does not participate in the intramolecular ligation as the 5ʹ HhaI site is absent in this form.
For these inverted samples, a second HhaI digestion fragment (nt147-1231, which is identical regardless of HBV DNA form) was used to quantify total HBV DNA.

Tu T., Zehnder B., Qu B, & Urban S. (2020). De novo synthesis of hepatitis B virus nucleocapsids is dispensable for the maintenance and transcriptional regulation of cccDNA. JHEP Reports, 3(1), 100195.

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Plasmid Barcode Sequencing Protocol

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Plasmid from the library was digested using SalI enzyme, and the digestion products were separated by gel electrophoresis, and cleaved backbone without 3′domain sequence was purified from gel. Seventy-five nanograms of the linear product containing the fragment and barcode was circularized using T4 DNA Ligase (NEB) 16°C overnight. The ligation product was treated with Lambda Exonuclease (NEB) and RecJF (NEB) for 16 h at 37°C (Balagurumoorthy et al. 2008 (link)). The remaining, circularized product was PCR amplified with primers containing Ion Torrent sites P1 & A. Concentration was determined by Bioanalyzer (Agilent) and sequenced on Ion Torrent using a 316 V2 chip (Thermo Fisher). Sequencing was conducted as 500-bp-long reads using an extended number of reagent flows in the 400 bp kit.

Davidsson M., Díaz-Fernández P., Torroba M., Schwich O.D., Aldrin-Kirk P., Quintino L., Heuer A., Wang G., Lundberg C, & Björklund T. (2018). Molecular barcoding of viral vectors enables mapping and optimization of mRNA trans-splicing. RNA, 24(5), 673-687.

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Plasmid Barcode Library Construction

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Plasmids from library 1 & 2 were digested using the SalI enzyme and the digestion products were separated by gel electrophoresis and cleaved backbone without 3′domain sequence was purified from gel. 75 ng of the linear product containing the fragment and barcode was circularized using T4 DNA Ligase (NEB) 16 °C over-night. The ligation product was treated with Lambda Exonuclease (NEB) and RecJF (NEB) for 16 hours at 37 °C36 (link). The remaining, circularized product was PCR amplified with primers containing Ion Torrent sites P1 & A. The concentration was determined by Bioanalyzer and sequenced on Ion Torrent using a 316 V2 chip (Thermo Fisher). Sequencing was conducted as 500 bp long reads using an extended number of reagent flows in the 400 bp kit.

Davidsson M., Diaz-Fernandez P., Schwich O.D., Torroba M., Wang G, & Björklund T. (2016). A novel process of viral vector barcoding and library preparation enables high-diversity library generation and recombination-free paired-end sequencing. Scientific Reports, 6, 37563.

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Recombinant ExonucleaseRecJ Assay

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RecJ_f (RecJ) used in this study is an exonuclease that catalyzes 5′ to 3′ degradation of ssDNA and is a recombinant of the native enzyme fused with maltose binding protein for increased water solubility. RecJ−SSBP test reactions used varying oligonucleotide concentrations, in units of RecJ and micrograms of SSBP, and contained the following reagents: RecJ_f [New England Biolabs (NEB)] (30 units/μL), ET SSB (Extreme Thermostable Single-Stranded DNA Binding Protein) (NEB) (0.5 μg/μL), and 1× NEBuffer 2 (NEB) [50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl₂, and 1 mM DTT (pH 7.9) at 25 °C]; RNase/DNase-free water was added for a total volume of 20 μL. Samples were incubated in a thermal cycler (Verti-96, Applied Biosystems) for 30 and 60 min at 37 °C followed by heat inactivation (20 min at 70 °C).

Jensen M, & Davis R. (2017). RecJ 5′ Exonuclease Digestion of Oligonucleotide Failure Strands: A “Green” Method of Trityl-On Purification. Biochemistry, 56(18), 2417-2424.

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Exonuclease Digestion for qPCR Cleanup

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Primers used for the reverse transcription (RP-TERRA and RP-Alu) and template oligonucleotides (OT and OT-Alu) could interfere with subsequent qPCR. To eliminate these oligonucleotides, the 5′ exonuclease RecJf was used. Phosphorothioate bonds incorporated at the 3′-terminal region of RP-TERRA and RP-Alu oligonucleotides prevented the digestion of the cDNA by RecJf. Nonspecific products may be rarely generated during qPCR, due to the presence of residual undigested template oligonucleotides. In this case, a second digestion step by RecJf may be necessary. Samples were heated at 95 °C for 3 min to denature DNA. Samples were immediately transferred to ice, and 1 µL of RecJf (New England Biolabs) was added. Samples were incubated at 37 °C for 1 h and 40 min to digest oligonucleotides, and then at 75 °C for 20 min to inactivate RecJf.

Le Berre G., Hossard V., Riou J.F, & Guieysse-Peugeot A.L. (2020). AP-TSS: A New Method for the Analysis of RNA Expression from Particular and Challenging Transcription Start Sites. Biomolecules, 10(6), 827.

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RNA Fragmentation and Adaptor Ligation

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RNA samples, typically 100 ng for DMSO and 1000 ng for NAI-N₃ or NAz-N₃ modified RNA, were first dried with a lyophilizer (Labconco) and brought back up 9 μl water. RNA was fragmented by adding 1 μl of Fragmentation Reagent (Ambion) at 70°C for 30 s and then stopped by adding 1 μl of RNA Fragmentation Stop Solution (Ambion) and moved to ice. RNA was desalted with a Zymo RNA clean and concentrator as above and again dried with a lyophilizer. End repair occurred by adding 5 μl of water, 2 μl of 5x PNK buffer (350 mM Tris–HCl pH 6.5, 50 mM MgCl₂, 25 mM DTT), 0.5 μl SUPERaseIn (Thermo Fisher Scientific), and 1.5 μl of T4 PNK (NEB) and 1.5 μl Fast AP (Thermo Fisher Scientific). These reactions were incubated at 37°C for 45 min. After end-repair, 1 μl of 2 μM 3′ pre-adenylated adaptor (3′biotin linker for DMSO or 3′dideoxycytosine linker for NAI-N₃ or NAz-N₃), 1 μl of 10× T4 RNA Ligase I buffer (NEB), 1 μl T4 RNA Ligase, High Concentration (NEB), 1 μl of 100 mM DTT and 6 μl of 50% PEG8000 were added. Ligation reactions were incubated at 25°C for 6 h. Excess, unligated adaptors were destroyed enzymatically by adding 3 μl of 10× NEB Buffer 2 (NEB), 2 μl Rec Jf (NEB), 1 μl 5′ deadenylase (NEB), 4 μl of water and incubate at 37°C for 1 h. Ligated RNA products were desalted and removed of enzymes with the Zymo RNA clean and concentrator.

Chan D., Feng C., England W.E., Wyman D., Flynn R.A., Wang X., Shi Y., Mortazavi A, & Spitale R.C. (2021). Diverse functional elements in RNA predicted transcriptome-wide by orthogonal RNA structure probing. Nucleic Acids Research, 49(20), 11868-11882.

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Photolyase-Mediated Repair of (6-4)PP Lesions

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Synthesized oligonucleotides with a single (6-4)PP were 3′ end-labeled as described above and purified through G25 gel filtration columns (GE Healthcare). About 0.1 pmol of radiolabeled substrates and 0.5 pmol of the same oligonucleotides unlabeled were incubated with 1.25 pmol of Escherichia coli (6-4)PP photolyase (Selby and Sancar 2012 (link)) in 50 µL of PLR buffer (50 mM Tris-HCl at pH7.5, 100 mM NaCl, 1 mM EDTA, 10 mM DTT) for 15 min or 2 h on ice with the irradiation of 18 mW/cm² 366 nm light from two black-light bulbs (F15T8-BLB, General Electric) filtered through one plate of glass. After phenol-chloroform extraction and ethanol precipitation, the oligonucleotides were incubated with 1 µL of RecJ_f (New England Biolabs) for 1 h at 37°C in 10 µL of 1× NEB2 buffer. Two microliters of reaction mixture was mixed with 10 µL of formamide loading buffer and incubated for 5 min at 95°C before separation with 20% denaturing sequencing gels. The gels were quantified by PhosphorImager (GE Healthcare).

Hu J., Adar S., Selby C.P., Lieb J.D, & Sancar A. (2015). Genome-wide analysis of human global and transcription-coupled excision repair of UV damage at single-nucleotide resolution. Genes & Development, 29(9), 948-960.

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Genomic DNA Extraction and Analysis

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All genomic DNA was extracted and purified using AxyPrep Blood Genomic DNA Miniprep Kit (Axygen) according to manufacturer’s instructions. DNA concentration was measured by Nanodrop-2000. For 2D agarose gel analysis, 10μg DNA was digested overnight at 37°C with 10U HinfI (Thermo Fisher), 10U RsaI (Thermo Fisher) and 2μg/mL RNase A (Takara). The reaction was terminated with EDTA and analyzed by 2D agarose gel electrophoresis. 30U RecJf (New England Biolabs) was added for removing 5' single-stranded DNA.
For internal gaps/nicks analysis, 5μg genomic DNA was digested overnight at 37°C with 5U HinfI (Thermo Fisher), 5U RsaI (Thermo Fisher) and 1μg/mL Ribonuclease A (RNase A, Takara) and purified with QIAquick PCR Purification kit (Qiagen). Purified DNA were digested with or without 200U Exonuclease III (New England Biolabs) overnight at 37°C, and then subjected to 0.7% agarose gel electrophoresis and in gel hybridization.

Zhang T., Zhang Z., Shengzhao G., Li X., Liu H, & Zhao Y. (2019). Strand break-induced replication fork collapse leads to C-circles, C-overhangs and telomeric recombination. PLoS Genetics, 15(2), e1007925.

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2E-CAS-seq: Improved RNA Sequencing

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The 2E-CAS-seq protocol was modified on the basis of CAS-seq by adding 4 U of 5′ deadenylase (NEB, Massachusetts, USA) and 9 U of RecJ_f (NEB, Massachusetts, USA) immediately after 3′ adapter ligation reaction. RecJf is an exonuclease that only digests single-strand DNA oligos with monophosphate at the 5′ end, but not the 3′ adapter bearing 5′ rApp modification, a 5′ deadenylase is used to remove the rApp modification from the 5′ end of the 3′ adapter, which convert the 3′ adapter to be a suitable substrate for RecJf digestion. The reaction mixture was incubated at 30 °C for 30 min followed by at 37 °C for 30 min. Then, the 3′ adapter ligation product was used for 5′ adapter ligation following the same method described in CAS-seq.

Yang Q., Li R., Lyu Q., Hou L., Liu Z., Sun Q., Liu M., Shi H., Xu B., Yin M., Yan Z., Huang Y., Liu M., Li Y, & Wu L. (2019). Single-cell CAS-seq reveals a class of short PIWI-interacting RNAs in human oocytes. Nature Communications, 10, 3389.

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Modified RNA Library Preparation

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The purified mRNA was subjected to icSHAPE library preparation as previously described with some modifications [19 (link)]. The in vivo modified RNA and unmodified RNA were biotinylated by copper-free click reaction, followed by fragmentation. The fragmented RNA was end repaired by incubation at 37 °C for 1 h with the following mix (70 mM Tris 7.0, 18 mM MgCl₂, 5 mM DTT, 4 U/μl RiboLock, 0.1 U/μl FastAP (Life Technology), 2 U/μl T4 PNK (NEB)) before 3′ adapter ligation with addition of 10 μl ligation mix (5 mM DTT, 0.5 μM 5′ adenylated and 3′-blocked linker (3′-bio for unmodified, 3′-ddc for modified), 0.66 U/μl T4 RNA ligase (NEB, M0437M), 15% PEG8000, 1X RNA ligase buffer) and incubation at 25 °C for extra 3 h. Then the excess adaptor was removed as described below. The purified RNA was incubated in the mix of 1 μl FastAP, 0.2 μl SSB (Promega), 0.8 μl RiboLock, and 1 μl 5′ Deadenylase (NEB) in 1× NEB buffer 2 (NEB) at 30 °C for 90 min. And then 1 μl RecJf (NEB) was added with another incubation at 37 °C for 1 h. The following procedures, including reverse-transcription, biotin-streptavadin enrichment, size selection of cDNA, circularization, and PCR amplification, were the same as described in the standard protocol.

Shi B., Zhang J., Heng J., Gong J., Zhang T., Li P., Sun B.F., Yang Y., Zhang N., Zhao Y.L., Wang H.L., Liu F., Zhang Q.C, & Yang Y.G. (2020). RNA structural dynamics regulate early embryogenesis through controlling transcriptome fate and function. Genome Biology, 21, 120.

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