Dna polymerase 1

Manufactured by New England Biolabs

Sourced in United States, China, United Kingdom

DNA polymerase I is an enzyme that catalyzes the synthesis of DNA. It plays a crucial role in DNA replication and repair processes.

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DNA polymerase (79 citations) E. coli DNA polymerase I (16 citations) DNA polymerase I Klenow fragment (14 citations) Klenow fragment of DNA polymerase I (8 citations) Escherichia coli DNA polymerase I (5 citations) DNA polymerase I Klenow (3 citations) Klenow fragment of E. coli DNA Polymerase I (2 citations) [α-32P]dATP and Klenow fragment of DNA polymerase I (1 citations)

67 protocols using dna polymerase 1

3C Assay Protocol for Chromatin Interaction Analysis

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Cells were fixed in 1% formaldehyde solution for 15 min and lysed with Hi-C lysis buffer (0.1%SDS; 50 mM HEPES–KOH, pH 7.5; 150 mM NaCl; 1 mM EDTA; 1% Triton X-100; 0.1% Sodium Deoxycholate). Permeable cells were treated with 0.5% SDS at 62 °C for 8 min and 1% Triton X-100 at 37 °C for 15 min. Next, chromatins were digested with MboI restriction enzyme (NEB) overnight at 37 °C and DNA Polymerase I (NEB) was added to incubate for 1 h. The digested DNA fragments were ligated with T4 DNA Ligase (NEB) for 4 h. Decrosslinking was performed with Proteinase K at 55 °C for 1 h. The ligated DNA fragments were purified with ethanol and sodium acetate. qRT-PCR was used to quantify 3C enrichment signals by normalizing them to the GAPDH locus.

Li M., Huang H., Wang B., Jiang S., Guo H., Zhu L., Wu S., Liu J., Wang L., Lan X., Zhang W., Zhu J., Li F., Tan J., Mao Z., Liu C., Ji J., Ding J., Zhang K., Yuan J., Liu Y, & Ouyang H. (2022). Comprehensive 3D epigenomic maps define limbal stem/progenitor cell function and identity. Nature Communications, 13, 1293.

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RNA-seq Library Construction and Analysis

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RNA-seq libraries were constructed as previously described⁴⁶. Briefly, seven days after transduction, cells were harvested and mRNA was purified from total RNA using oligo(dT) Dynabeads (Invitrogen). First-strand cDNA was synthesized using the SuperScript VILO cDNA Synthesis Kit (Invitrogen) and second-strand cDNA was synthesized using DNA polymerase I (New England Biolabs). cDNA was purified using Agencourt AMPure XP beads (Beckman Coulter). Purified cDNA was treated with Nextera transposase (Illumina) for 5 min at 55 °C to simultaneously fragment and insert sequencing primers into the double-stranded cDNA. Transposase activity was halted using QG buffer (Qiagen) and fragmented cDNA was purified on AMPure XP beads. Indexed sequencing libraries were PCR-amplified and sequenced for 50-bp paired-end reads on an Illumina HiSeq 2000 instrument at the Duke Genome Sequencing Shared Resource and for 75 paired-end reads on an Illumina MiSeq. Reads were trimmed to 50 bp and aligned to the delivered lentiviral vector were removed from analysis using Bowtie2⁴⁷. Filtered reads were then aligned to human RefSeq transcripts using Bowtie2. Statistical analysis, including multiple hypothesis testing, on three independent biological replicates was performed using DESeq⁴⁸.

Thakore P.I., D’Ippolito A.M., Song L., Safi A., Shivakumar N.K., Kabadi A.M., Reddy T.E., Crawford G.E, & Gersbach C.A. (2015). Highly Specific Epigenome Editing by CRISPR/Cas9 Repressors for Silencing of Distal Regulatory Elements. Nature methods, 12(12), 1143-1149.

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RNA-Seq Library Preparation and Sequencing

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According to the manufacturer’s manual, sequencing libraries were generated using NEBNext Ultra™ RNA Library Prep Kit for Illumina (NEB, USA). Amount of 3 μg RNA per sample was used to purify mRNA using poly-T oligo attached magnetic beads and then the purified mRNA was interrupted into short fragments (about 200 bp) by the fragmentation buffer. First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H). After adding buffer, dNTPs, DNA polymerase I (New England Biolabs) and RNase H (Invitrogen), the second strand cDNA synthesis was subsequently performed using DNA polymerase I and RNase H. After adenylation of 3’ ends of DNA fragments, NEBNext Adapter was ligated to prepare for hybridization. The cDNA fragments with 150–200 bp in length were selected for PCR amplification to create cDNA libraries. The library preparations were sequenced on an Illumina Hiseq 2500 platform and generated 125 bp paired-end reads.

Chu Q., Zhou B., Xu F., Chen R., Shen C., Liang T., Li Y, & Schinckel A.P. (2017). Genome-wide differential mRNA expression profiles in follicles of two breeds and at two stages of estrus cycle of gilts. Scientific Reports, 7, 5052.

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Transcriptome Library Construction and Sequencing

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The cDNA library was constructed using the NEBNext® Ultra^TM RNA kit according to the manufacturer’s instructions. In brief, 45 μg of total RNA from each sample was prepared at a concentration of approximately 1,500 ng·μL⁻¹. Poly (A) mRNAs were enriched using oligo (dT) beads and interrupted into short fragments with fragmentation buffer. First-strand cDNA was synthesized using hexamer primers and reverse transcriptase (Invitrogen). The second-strand cDNA was then synthesized using buffer, dNTPs, RNaseH (Invitrogen), and DNA polymerase I (New England BioLabs).
For the construction of the two paired-end libraries, the double-stranded fragments were then purified using a QiaQuick PCR extraction kit and resolved with EB buffer to finish the end reparation, and were connected using sequencing adaptors. The successfully connected fragments were then subjected to sequencing using the Illumina HiSeqTM 2500 sequencing platform. The raw reads were initially processed by removing adaptor sequences and low quality reads using an in-house Perl software, where the average proportion of clean reads in each sample was approximately 91.5%. the low quality reads here refers to reads whose ratios of unknown bases are higher than 5%, or ratios of low quality bases (base quality≤10) are higher than 20%.

Dong B., Wu B., Hong W., Li X., Li Z., Xue L, & Huang Y. (2017). Transcriptome analysis of the tea oil camellia (Camellia oleifera) reveals candidate drought stress genes. PLoS ONE, 12(7), e0181835.

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Illumina Sequencing and De Novo Transcriptome Assembly

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For Illumina sequencing, the poly(A) mRNA was fragmented into small pieces. First-strand cDNA synthesis was conducted using random hexamer primers and reverse transcriptase (Invitrogen, Shanghai, China). Second-strand cDNA was synthesized using RNase H (Invitrogen) together with DNA polymerase I (New England BioLabs, Ipswich, MA, USA). We further constructed 18 cDNA libraries and sequenced the cDNA on an Illumina HiSeq 2000 platform according to the provided manual. For de novo assembly, first, the duplicated reads and low-quality reads were removed. This yielded 617,356,914 clean paired-end reads (Table 1). Unigenes were then de novo assembled using Trinity [55 (link)] through all of the seven RNA-Seq samples in the wild-type JI2822 background, respectively. We then used CAP3 to further assemble overlapping regions from a pool of all contigs from each of the RNA-Seq assemblies [56 (link)]. The resulting cap3 contigs and singlets were combined together and those that were smaller than 200 nucleotides were eliminated. A total of 192,977,028 clean reads were used during the assembly and this yielded 87,137 unigenes. The distribution of the de novo assembled unigenes is presented in Figure 1.

Jiao K., Li X., Guo W., Su S, & Luo D. (2017). High-Throughput RNA-Seq Data Analysis of the Single Nucleotide Polymorphisms (SNPs) and Zygomorphic Flower Development in Pea (Pisum sativum L.). International Journal of Molecular Sciences, 18(12), 2710.

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6mA Methylation Profiling in Oxytricha

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Genomic DNA was isolated from vegetative Oxytricha cells using the Nucleospin Tissue Kit (Takara Bio USA, Inc.). DNA was sheared into 150bp fragments using a Covaris LE220 ultra-sonicator (Covaris). Samples were gel-purified on a 2% agarose-TAE gel, blunted with DNA polymerase I (New England Biolabs), and purified using MinElute spin columns (QIAGEN). The fragmented DNA was dA-tailed using Klenow Fragment (3′ -> 5′ exo-) (New England Biolabs) and ligated to Illumina adaptors following manufacturer’s instructions. Subsequently, 2.2μg of adaptor-ligated DNA containing 6mA was immunoprecipitated using an anti-N6-methyladenosine antibody (Cedarlane Labs) conjugated to Dynabeads Protein A (Invitrogen). The anti-6mA antibody is commonly used for RNA applications, but has also been demonstrated to recognize 6mA in DNA (Fioravanti et al., 2013 (link); Xiao and Moore, 2011 ). The immunoprecipitated and input libraries were treated with proteinase K, extracted with phenol:chloroform, and ethanol precipitated. Finally, they were PCR-amplified using Phusion Hot Start polymerase (New England Biolabs) and used for Illumina sequencing.

Beh L.Y., Debelouchina G.T., Clay D.M., Thompson R.E., Lindblad K.A., Hutton E.R., Bracht J.R., Sebra R.P., Muir T.W, & Landweber L.F. (2019). Identification of a DNA N6-adenine methyltransferase complex and its impact on chromatin organization. Cell, 177(7), 1781-1796.e25.

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Differential Expression Analysis of CircRNAs

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To identify differentially expressed circRNAs, the left lungs of sham (n = 3) and CLP (n = 3) mice were used for high-throughput sequencing (Nanjing Decode Genomics Center, Nanjing, China) (Figure 1). Total RNA was extracted and ribosomal RNA was removed. A strand-specific RNA library was constructed. RNAs were digested into 200–500 bp fragments and then used as templates to synthesize cDNAs in the presence of a six-base random primer, dNTPs, RNase H (Thermo Scientific, USA), DNA polymerase I (New England Biolabs Inc., USA), and buffer (Thermo Scientific, USA). The PCR products were purified using the QiaQuick PCR kit (QIAGEN, GER) and end-repaired, followed by A-tail addition and sequencing-linker ligation. Fragment sizes were determined by agarose gel electrophoresis and then PCR amplification was performed. The sequenced library was used for Sanger sequencing by adopting a double-ended sequencing strategy.Figure 1

Flow chart.

Figure 1

Based on the TopHat comparison results, HTSeq software was used to compare the number of reads mapped to each gene by adopting default parameters, and the results were used to calculate the expression level of each gene. To identify differentially expressed genes, further analysis of different specimens (groups) was carried out using edgeR software (http://www.bioconductor.org/packages/release/bioc/html/edgeR.html).

Yuan C., Gu J., Wu J., Yin J., Zhang M., Miao H, & Li J. (2020). Circular RNA expression in the lungs of a mouse model of sepsis induced by cecal ligation and puncture. Heliyon, 6(7), e04532.

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Minigene Construction for ECS Study

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The minigene gIQECS was generated by ligating a SpeI-XhoI genomic DNA fragment digested from mouse bacterial artificial chromosome (Library Plates 481, ResGen, USA, CA) into the modified pRK5 vector digested by XbaI and SalI. The 4952 bp genomic DNA contains the putative ECS, intermediate sequence and exon 41. The modified PRK5 vector was generously provided by Dr Miyoko Higuchi from Max-Planck Institute for Medical Research, Germany. Subsequently, in the same vector the ECS sequence was deleted from by overlapping PCR to produce the gIQΔECS minigene and the short IQECS minigene that supports the expression of only the RNA duplex was constructed by PCR prior to cloning into the modified pRK5 vector. All restriction enzymes, DNA Polymerase I, Large (Klenow) Fragment and T4 DNA ligase were purchased from New England Biolabs, USA and used per manufacturer's instruction.

Huang H., Kapeli K., Jin W., Wong Y.P., Arumugam T.V., Koh J.H., Srimasorn S., Mallilankaraman K., Chua J.J., Yeo G.W, & Soong T.W. (2018). Tissue-selective restriction of RNA editing of CaV1.3 by splicing factor SRSF9. Nucleic Acids Research, 46(14), 7323-7338.

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Illumina Sequencing of Silkworm Larvae

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For Illumina sequencing, equivalent quantities of total RNA were isolated from the three larvae and BmPSG were pooled. After poly (A) mRNA was purified and fragmented into smaller fragments, random hexamer primers and reverse transcriptase (Invitrogen, Life Technologies, Carlsbad, CA, USA) were used to carry out first strand cDNA synthesis. Second strand cDNA synthesis was performed with RNase H (Invitrogen) and DNA polymerase I (New England BioLabs, Beijing, China). We constructed a cDNA library with average insert sizes of 200–500 bp and conducted cDNA sequencing using the Illumina HiSeq™ 2000 system according to the manufacturer’s protocols, with a read length of 50 bp. RNA-Seq Quantification analyses used two independent cDNA libraries, and were constructed for the two organs in parallel according to the RNA-Seq protocol. The RNA-seq sequencing data were made available to BGI (BGI, Shenzhen, China).

Wang H., Wang L., Wang Y., Tao H., Yin W., SiMa Y., Wang Y, & Xu S. (2015). High yield exogenous protein HPL production in the Bombyx mori silk gland provides novel insight into recombinant expression systems. Scientific Reports, 5, 13839.

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R-loop Mapping with DRIP Protocol Modifications

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R-loop mapping was performed following the DRIP protocol^{50 (link)} with some modifications. PATC53 cells were treated with 1 µM PRMTi or DMSO as control. One and three days later, DNA was extracted as described^{76 (link)}, but genome fragmentation was conducted by shearing via sonication using a Diagenode Bioruptor (12 cycles, High, 15’ON 90’OFF). As sonication degrades the single-stranded looped out DNA strand of R-loops, immunoprecipitation with S9.6 enriches mostly for two-stranded RNA: DNA hybrids. To build sequencing libraries, hybrids were transformed back into double-stranded DNA via a second-strand DNA synthesis step using E. coli RNase H1, DNA ligase, DNA polymerase I (New England Biolabs) and a dNTP mix in which dUTP was used instead of dTTP. After checking the quality of the immunoprecipitation by qPCR, the DNA was built into strand-specific sequencing libraries with a UDG DNA glycosylase step before the PCR amplification step to ensure strand specificity^{76 (link)}. Library quality was checked on an Agilent BioAnalyzer and sequencing performed on an Illumina HiSeq4000 instrument. Mapping was performed on two independent biological replicates.

Giuliani V., Miller M.A., Liu C.Y., Hartono S.R., Class C.A., Bristow C.A., Suzuki E., Sanz L.A., Gao G., Gay J.P., Feng N., Rose J.L., Tomihara H., Daniele J.R., Peoples M.D., Bardenhagen J.P., Geck Do M.K., Chang Q.E., Vangamudi B., Vellano C., Ying H., Deem A.K., Do K.A., Genovese G., Marszalek J.R., Kovacs J.J., Kim M., Fleming J.B., Guccione E., Viale A., Maitra A., Emilia Di Francesco M., Yap T.A., Jones P., Draetta G., Carugo A., Chedin F, & Heffernan T.P. (2021). PRMT1-dependent regulation of RNA metabolism and DNA damage response sustains pancreatic ductal adenocarcinoma. Nature Communications, 12, 4626.

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