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.
Dutp solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
The DUTP Solution is a laboratory reagent used in various molecular biology applications. It is a buffered solution containing the nucleotide deoxyuridine triphosphate (dUTP). The DUTP Solution is designed to be used as a component in specific experimental protocols, but its core function is to provide a source of dUTP for those applications.
Automatically generated - may contain errors
Lab products found in correlation
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (11 mentions)
E coli dna polymerase 1, by New England Biolabs (10 mentions)
Rnase h, by New England Biolabs (10 mentions)
Magnesium rna fragmentation module, by New England Biolabs (7 mentions)
Novaseq 6000, by Illumina (7 mentions)
Udg enzyme, by New England Biolabs (4 mentions)
Trizol, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Dynabeads oligo dt, by Thermo Fisher Scientific (3 mentions)
Dynabeads oligo, by Thermo Fisher Scientific (3 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Heat labile udg enzyme, by New England Biolabs (2 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (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)
Rnaiso plus reagent, by Takara Bio (1 mentions)
M6a antibody, by Synaptic Systems (1 mentions)
Dynabeads antibody coupling kit, by Thermo Fisher Scientific (1 mentions)
M6a specific antibody, by Synaptic Systems (1 mentions)
13 protocols using dutp solution
1
RNA Isolation, Purification, and Sequencing Protocol
2
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.
3
RNA-seq Library Preparation Protocol
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Total RNA was extracted using RNAiso Plus reagent (Takara, cat. no. 9108) according to the manufacturer's instructions. The quality of the extracted total RNA was detected by NanodropTM spectrophotometer (Thermo Fisher Scientific Inc) and agarose gel electrophoresis. Then, mRNA was purified from 2 μg total RNA by using Dynabeads Oligo (dT) (Thermo Fisher Scientific) with two rounds of purification and fragmented with divalent cations under elevated temperature. Then the cleaved RNA fragments were reverse-transcribed to create the cDNA by SuperScript II Reverse Transcriptase (Invitrogen, cat. no. 1896649), which were next used to synthesize U-labeled second-stranded DNAs with E. coli DNA polymerase I (NEB, cat. no. m0209), RNase H (NEB, cat. no. m0297) and dUTP Solution (Thermo Fisher Scientific, cat. no. R0133). After the heat-labile UDG enzyme (NEB, cat. no. m0280) treatment of the U-labeled second-stranded DNAs, the ligated products were amplified with PCR. The average insert size for the final cDNA librarys were 300 ± 50 bp. At last, we performed the 2 × 150 bp paired-end sequencing (PE150) on an Illumina NovaSeq 6000 (LC-Bio Technology CO, Ltd) following the vendor's recommended protocol.
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
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.
10
Illumina-based Transcriptome Profiling
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Poly(A) RNA was purified from 0.4 μg total RNA using Dynabeads Oligo(dT)25–61005 (Thermo Fisher, USA). The poly(A) RNA was fragmented into small pieces using Magnesium RNA Fragmentation Module (NEB, USA). The cleaved RNA fragments were reverse-transcribed into cDNA by SuperScript II Reverse Transcriptase (Invitrogen), which was subsequently used to synthesize U-labelled second-stranded DNAs with E. coli DNA polymerase I (NEB), RNase H (NEB) and dUTP Solution (Thermo Fisher). Size selection was performed with AMPure XP beads (Beckman Coulter, USA). After the heat-labile UDG enzyme (NEB) treatment of the U-labelled second-stranded DNAs, the ligated products were amplified with PCR. The average insert size for the final cDNA library was 300 ± 100 bp. The 2 × 150 bp pair-end sequencing for mRNA were performed on Illumina Hiseq 2500 (Genechem Technology Co. Ltd., Shanghai, China).
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