Sample RNA was isolated and purified using TRIzol (cat. 15596018; Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The quantity and purity of total RNA were then quality controlled using the NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE, USA) and the integrity of RNA was checked using a Bioanalyzer 2100 (Agilent Technologies Co. Ltd., Palo Alto, CA, USA). Concentrations >50 ng/µL, RIN value >7.0, and total RNA >1 µg were sufficient for downstream experiments. The RNA was subsequently reverse transcribed to cDNA using SuperScript™ II reverse transcriptase (Invitrogen, cat. 1896649, USA). cDNA was purified using E. coli DNA polymerase I (NEB, cat. m0209, USA), RNaseH (NEB, cat. m0297, USA), and dUTP solution (Thermo Fisher, cat. R0133, USA). Following this, the junction was ligated to the A-tailed fragment DNA and further purification was performed. Finally, amplification was conducted by PCR, and 2×150 bp double-end sequencing (PE150) was performed using Illumina Novaseq™ 6000 (LC-Bio Technology Co., Ltd., Hangzhou, China). The amplification conditions were as follows: Initial denaturation at 95 ℃ for 3 min; denaturation at 98 ℃ for 15 s, annealing at 60 ℃ for 15 s, extension at 72 ℃ for 30 s for eight cycles; and final extension at 72 ℃ for 5 min.
E coli dna polymerase 1
Manufactured by New England Biolabs
Sourced in United States, United Kingdom
E. coli DNA polymerase I is an enzyme responsible for the replication and repair of DNA. It catalyzes the addition of nucleotides to the 3' end of a DNA strand, using a DNA template. The enzyme possesses both 5'-3' polymerase and 3'-5' exonuclease activities, allowing it to synthesize new DNA as well as proofread and correct errors during the process.
Automatically generated - may contain errors
Lab products found in correlation
Dutp solution, by Thermo Fisher Scientific (10 mentions)
Rnase h, by New England Biolabs (10 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (9 mentions)
Novaseq 6000, by Illumina (6 mentions)
Trizol reagent, by Thermo Fisher Scientific (6 mentions)
Magnesium rna fragmentation module, by New England Biolabs (5 mentions)
Udg enzyme, by New England Biolabs (4 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Dynabeads oligo, by Thermo Fisher Scientific (2 mentions)
Heat labile udg enzyme, by New England Biolabs (2 mentions)
Dynabeads oligo dt, by Thermo Fisher Scientific (2 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (2 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (1 mentions)
Ultralow v2, by Tecan (1 mentions)
Nebuffer 2, by New England Biolabs (1 mentions)
T4 dna polymerase, by New England Biolabs (1 mentions)
Dynabeads mrna purification kit, by Thermo Fisher Scientific (1 mentions)
Anti m6a antibody, by Synaptic Systems (1 mentions)
Novaseq 6000 platform, by Illumina (1 mentions)
16 protocols using e coli dna polymerase 1
1
RNA Isolation, Purification, and Sequencing Protocol
2
Enzymatic Fragmentation and Adapter Ligation
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Example 4
Intact genomic DNA was fragmented in a 15 ul reaction containing 2 mU HL-dsDNase (ArcticZymes), 6 U E. coli DNA Polymerase I (NEB), 1.5 U T4 DNA Polymerase, 1×NEBuffer 2 (NEB) and 0.2 mM dNTPs under the following temperature profile: 25 C for 15 min, 65 C for 15 min, 4 C hold. The NuGEN Ultralow v2 ligation and PCR components were used to perform ligation and PCR as follows. Ligation was performed by adding 3 ul of Ligation Adaptor Mix, 5 ul of Ligation Buffer Mix, and 2 ul of Ligation Enzyme Mix for a total of 25 ul. After the standard ligation incubation steps of 25 C for 30 min, 70 C for 10 min, and 4 C hold, PCR components were added directly to the ligation reaction, without bead purification. 25.5 ul of Amp Buffer Mix, 2.5 ul of Amp Primer Mix, 2 ul of Amp Enzyme Mix, and 45 ul of water were added to prepare a 100 ul PCR reaction. After 9 cycles of PCR following the cycling conditions described in the Ultralow user guide the PCR products were bead purified and analyzed by Bioanalyzer.
3
m6A Methylome Profiling in A375 Cells
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Total RNA was isolated and purified using TRIzol reagent (Invitrogen, USA) from indicated A375 cells. The RNA amount and purity of each sample was quantified using NanoDrop ND-1000 (NanoDrop, USA), and the RNA integrity was assessed by Bioanalyzer 2100 (Agilent, USA). The mRNA was further purified using Dynabeads mRNA Purification Kit (61006, Invitrogen, USA). After fragmentation with Magnesium RNA Fragmentation Module (NEB, USA), the anti-m6A antibody (202003, Synaptic Systems, Germany) was used for immunoprecipitated. The IP RNA was reverse-transcribed to cDNA by SuperScript™ II Reverse Transcriptase (Invitrogen, USA), which was next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I (NEB, USA), RNase H (NEB, USA) and dUTP Solution (Thermo Fisher, USA). At last, we performed paired-end sequencing (PE150) on an Illumina Novaseq™ 6000 platform (LC-Bio, Hangzhou, China) or MeRIP-qPCR analysis.
4
Illumina-based RNA-seq protocol
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Poly(A) RNA was selected from 1 μg total RNA in each sample using Dynabeads Oligo (dT)25 (Thermo Fisher) through two rounds of purification and fragmented into small pieces using Magnesium RNA Fragmentation Module (New England Biolabs) under 94°C for 5–7 min. The cleaved RNA fragments were firstly reverse transcribed using SuperScript™ II Reverse Transcriptase (Invitrogen). Then the second strand of cDNA was synthesized using E. coli DNA polymerase I (New England Biolabs), RNase H (New England Biolabs), and marked with dUTP Solution (Thermo Fisher). Subsequently, after end polishing, A-tailing, adapter ligation, and size selection (using AMPureXP beads), the dUTP-marked strand was selectively degraded by Uracil-DNA-Glycosylase (UDG). The remaining strand was amplified with PCR (95°C for 3 min, 15 cycles of “98°C for 15 s, 60°C for 15 s, and 72°C for 30 s”, and 72°C for 5 min) to generate a cDNA library with 300 ± 50 bp. Libraries were multiplexed in equimolar concentrations and sequenced using an Illumina Novaseq™ 6000 with 2 × 150 bp paired-end sequencing (LC-Bio Technology CO., Ltd.) following the vendor’s recommended protocol.
5
RNA Extraction and Sequencing
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Total RNA of each sample was extracted and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), followed by fragmentation using Magnesium RNA Fragmentation Module (NEB, USA), and synthesis of U-labeled second-stranded DNAs using SuperScript™ II Reverse Transcriptase (Invitrogen, USA), E. coli DNA polymerase I (NEB, USA), RNase H (NEB, USA) and dUTP Solution (Thermo Fisher, USA). After single- or dual-index adapters were ligated to the fragments, the U-labeled second-stranded DNAs were treated with heat-labile UDG enzyme (NEB, USA), and the ligated products were amplified with PCR. Sequencing data were generated on Illumina Novaseq™ 6000 (LC Sciences, USA) with 2 × 150 bp paired-end sequencing (PE150) following the vendor's recommended manual.
6
m6A-Seq RNA Enrichment and Sequencing
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The Poly (A) RNA was purified from 50 μg total RNA using Dynabeads Oligo (dT) (Thermo Fisher, CA, USA) through two rounds of purification. The fragmented RNA was premixed with the Dynabeads Antibody Coupling Kit (Thermo Fisher, CA, USA) and m6A antibody (Synaptic Systems, Germany) in IP buffer and incubated for 2 h at 4 °C. The IP RNA was reverse-transcribed to synthesize the cDNA using the SuperScript™ II Reverse Transcriptase (Invitrogen, USA). Then the two-strand synthesis was performed with RNase H (NEB, USA) using the E. coli DNA polymerase I (NEB, USA) to synthesize U-labeled second-stranded DNAs. At the same time, the dUTP Solution (Thermo Fisher, CA, USA) was incorporated into the two strandsand the size of the fragment was screened and purified using the AMPureXP beads. The U-labeled second-stranded DNAs were digested using a heat-labile UDG enzyme (NEB, USA), then amplified by PCR to form a cDNA library of 300 ± 50 BP. At last, the 2 × 150 bp paired-end sequencing (PE150) was performed on an Illumina Novaseq™ 6000 (LC-Bio Technology CO., Ltd., Hangzhou, China) following the vendor's recommended protocol.
7
m6A-seq Library Preparation
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The total RNA was fragmented and then incubated with m6A-specific antibody (No. 202003, Synaptic Systems, Germany) in IP buffer (50 mM Tris–HCl, 750 mM NaCl, and 0.5% Igepal CA-630) for 2 h at 4°C. Then, the IP RNA was reverse-transcribed to cDNA, and U-labeled second-stranded DNAs were synthesized using cDNA with E. coli DNA polymerase I, RNase H (NEB, United States), and dUTP Solution (Thermo Fisher Scientific, United States). Then, the strands were prepared with A-base and ligated to the indexed adapters containing a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. AMPureXP beads were used to screen fragments of the right size. The U-labeled second-stranded DNAs treated with heat-labile UDG enzyme (NEB, United States) were next amplified by PCR to generate a cDNA library with an average insert size of 300 ± 50 bp. Finally, 2 × 150 bp paired-end sequencing (PE150) was performed on an Illumina NovaseqTM 6000 (LC-BioTechnology Co., Ltd., Hangzhou, China), following the manufacturer’s recommended protocol.
8
Illumina-based RNA-seq protocol
9
ChIP-seq Analysis of Oocyte Histone
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For ChIP-seq analyses, 364 and 140 NT oocytes were used for histone B4 and H3 library preparation, respectively. Antibodies used include a rabbit polyclonal B4 antibody (Ohsumi et al., 1993 (link)) and a rabbit polyclonal anti-histone H3 antibody (ab1791, Abcam). DNA fragments obtained from ChIP pulldowns were subjected to E. coli DNA polymerase I (New England Biolabs, M0209) treatment before end repair reaction. The samples were then amplified by following the library preparation protocol described for RNA-seq (20 PCR cycles). The ChIP-seq libraries were sequenced on an Illumina HiSeq 2000.
10
Reverse Transcription and cDNA Library Preparation
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First, the cleaved RNA fragments were reverse-transcribed to create the cDNA by SuperScript™ II Reverse Transcriptase (Invitrogen, cat. 1896649, USA). Second, it was used to synthesize U-labeled second-stranded DNAs with E. coli DNA polymerase I (NEB, cat.m0209, USA), RNase H (NEB, cat.m0297, USA), and dUTP Solution (Thermo Fisher, cat.R0133, USA). Third, an A-base was added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. After the heat-labile UDG enzyme (NEB, cat.m0280, USA) treatment of the U-labeled second-stranded DNAs, the ligated products were amplified with PCR as conventional conditions. Finally, the average insert size for the final cDNA library was 300±50 bp.
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