For WGBS, 1 × 106 cells were collected for each sample. Briefly, DNAs were extracted using Allprep DNA/RNA/Protein Mini Kit (Qiagen, 80004) following the manufacturer's instruction. The qualities of total DNAs were determined by NanoDrop 2000 and agarose electrophoresis. High‐quality DNA sample (1 µg) spiked with 26 ng unmethylated lambda DNA (Promega, D1521) was fragmented into ≈250‐bp fragments using S220 Focused‐ultrasonicator (Covairs). After end‐repairing and dA‐tailing using 5× ER/A‐Tailing Enzyme Mix (Enzymatics, Y9420L), fragmented DNAs were ligated using cytosine‐methylated barcodes and treated with bisulfite with EZ DNA Methylation‐Gold Kit (Zymo, D5006). The WGBS libraries were then generated by PCR. Subsequently, sequencing was performed in Illumina Novaseq 6000 sequencer using 150 bp pair‐end protocol.
Er a tailing enzyme mix
Manufactured by Qiagen
Sourced in United States
The 5× ER/A‐Tailing Enzyme Mix is a reagent used in molecular biology applications. It is designed to perform end-repair and A-tailing of DNA fragments in a single reaction. The mix contains enzymes that convert 3' overhangs and 5' phosphates into blunt ends, and then adds a single adenine (A) nucleotide to the 3' ends. This prepares the DNA fragments for ligation with complementary vectors or adapters.
Automatically generated - may contain errors
Lab products found in correlation
Unmethylated lambda dna, by Promega (1 mentions)
Allprep dna rna protein mini kit, by Qiagen (1 mentions)
Novaseq 6000 sequencer, by Illumina (1 mentions)
Ez dna methylation gold kit, by Zymo Research (1 mentions)
User enzyme mix, by New England Biolabs (1 mentions)
Ampure xp bead, by Illumina (1 mentions)
Nextseq platform, by Illumina (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
2 protocols using er a tailing enzyme mix
1
Whole-Genome Bisulfite Sequencing Protocol
2
NGS Library Preparation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
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All NGS samples were prepared
using the 5× ER/A-tailing Enzyme Mix (Enzymatics, USA) and WGS
ligase (Enzymatics, USA) without deviating from the manufacturer’s
protocol. DNA was first mixed with the ER/A-tailing enzyme mix and
incubated at 20 °C for 30 min and 65 °C for 30 min. Sequencing
adaptor (10 μL) from NEBNext Multiplex Oligos for Illumina (NEB,
USA) and WGS ligase were added to the A-tailed product. After ligation
for 30 min at 23 °C, 3 μL of USER enzyme mix (New England
BioLabs, USA) was directly mixed into the ligation tube and incubated
for 30 min at 37 °C. The USER cleaved product was then purified
using Agencourt AMPure XP beads with a 1.2× excess volume of
USER cleaved product (Beckman-Coulter, USA). The products were amplified
with either PCR or RPA using the index primer pairs provided in the
NEBNext Multiplex Oligos for Illumina kit. The product was purified
using AMPure XP beads with a 1.2× excess volume of AMPure XP
beads and sequenced using the Illumina NextSeq platform.
using the 5× ER/A-tailing Enzyme Mix (Enzymatics, USA) and WGS
ligase (Enzymatics, USA) without deviating from the manufacturer’s
protocol. DNA was first mixed with the ER/A-tailing enzyme mix and
incubated at 20 °C for 30 min and 65 °C for 30 min. Sequencing
adaptor (10 μL) from NEBNext Multiplex Oligos for Illumina (NEB,
USA) and WGS ligase were added to the A-tailed product. After ligation
for 30 min at 23 °C, 3 μL of USER enzyme mix (New England
BioLabs, USA) was directly mixed into the ligation tube and incubated
for 30 min at 37 °C. The USER cleaved product was then purified
using Agencourt AMPure XP beads with a 1.2× excess volume of
USER cleaved product (Beckman-Coulter, USA). The products were amplified
with either PCR or RPA using the index primer pairs provided in the
NEBNext Multiplex Oligos for Illumina kit. The product was purified
using AMPure XP beads with a 1.2× excess volume of AMPure XP
beads and sequenced using the Illumina NextSeq platform.
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