Purified ChIP DNA from a standard ChIP for H3K4me3 on PBMCs was measured using a Qubit fluorometer (Life Technologies) and then diluted in 10mM Tris-Cl pH 8.5 supplemented with 0.1% Tween-20 to 100 pg, 10 pg, or 2 pg total DNA. The tagmentation reaction was performed for 5 minutes at 55°C in a 10 μl reaction containing diluted DNA, 5 μl 2x tagmentation buffer (Illumina) and 1 μl (for 100 pg DNA) or 0.5 μl (for 10 pg and 2 pg) 1:10 diluted Nextera Tag DNA Enzyme (diluted in precooled TE/50% glycerol). The tagmented DNA was amplified with the Nextera DNA Sample Prep Kit (Illumina) according to the manufacturer’s instructions with the following program: 72°C 5 min, 98°C 30 s, 14 cycles of 98°C 10 s 63°C 30 s 72°C 30 s, and a final elongation at 72°C for 1 min. Libraries were purified using SPRI AMPure XP beads with a beads-to-sample ratio of 1.5:1. Purified ChIP DNA or deproteinized input DNA from K562 ChIP was prepared as for PBMCs with slight modifications: 5 ng of ChIP DNA was taken for the tagmentation reaction using 0.5 μl of a 1:10 diluted Tn5 transposase in a 5 μl reaction at 55°C for 5 minutes. DNA was purified with the MinElute kit (Qiagen) and amplified with the Kapa HiFi HotStart ReadyMix (Kapa Biosystems).
Nextera tag dna enzyme
Manufactured by Illumina
The Nextera Tag DNA Enzyme is a laboratory equipment product designed for preparing DNA samples for sequencing. It is used to fragment DNA and add adapter sequences, which are necessary for the subsequent steps in the DNA sequencing process. The core function of the Nextera Tag DNA Enzyme is to prepare DNA samples for downstream analysis and sequencing.
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2 protocols using nextera tag dna enzyme
1
Tagmentation and Library Preparation for ChIP-seq
2
Tagmentation and Library Preparation for ChIP-seq
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Purified ChIP DNA from a standard ChIP for H3K4me3 on PBMCs was measured using a Qubit fluorometer (Life Technologies) and then diluted in 10mM Tris-Cl pH 8.5 supplemented with 0.1% Tween-20 to 100 pg, 10 pg, or 2 pg total DNA. The tagmentation reaction was performed for 5 minutes at 55°C in a 10 μl reaction containing diluted DNA, 5 μl 2x tagmentation buffer (Illumina) and 1 μl (for 100 pg DNA) or 0.5 μl (for 10 pg and 2 pg) 1:10 diluted Nextera Tag DNA Enzyme (diluted in precooled TE/50% glycerol). The tagmented DNA was amplified with the Nextera DNA Sample Prep Kit (Illumina) according to the manufacturer’s instructions with the following program: 72°C 5 min, 98°C 30 s, 14 cycles of 98°C 10 s 63°C 30 s 72°C 30 s, and a final elongation at 72°C for 1 min. Libraries were purified using SPRI AMPure XP beads with a beads-to-sample ratio of 1.5:1. Purified ChIP DNA or deproteinized input DNA from K562 ChIP was prepared as for PBMCs with slight modifications: 5 ng of ChIP DNA was taken for the tagmentation reaction using 0.5 μl of a 1:10 diluted Tn5 transposase in a 5 μl reaction at 55°C for 5 minutes. DNA was purified with the MinElute kit (Qiagen) and amplified with the Kapa HiFi HotStart ReadyMix (Kapa Biosystems).
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