Five million cells from MV4;11 and MOLM13 were washed in PBS and resuspend in a hypotonic buffer (50 mM KCl, 10 mM MgSO4, 5 mM HEPES, 0.05% NP-40, 1 mM PMSF, 3 mM DTT) to generate intact nuclei and washed three times in a nuclear wash buffer (10 mM Tris HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2). 50,000 nuclei per cell line were used for the transposition reaction. Nuclei were incubated in 1X TD Buffer (Illumina) and 2.5 μM Transposase enzyme (Illumina) for 30 minutes at 37 °C. Transposed DNA was then extracted from nuclei using the MiniElute PCR purification Kit (Qiagen). Transposed DNA fragments were amplified using NEBNext® High-Fidelity PCR kit (NEB) with a universal Nextera primer and a unique indexing primer and sequenced using NextSeq550 (Illumina) obtaining paired end reads (R1: 75bp; R2: 75bp) with 57 million and 48 million reads obtained for the MOLM13 and MV4;11 cell lines, respectively.
Nebnext high fidelity pcr kit
Manufactured by New England Biolabs
The NEBNext® High-Fidelity PCR kit is a reagent kit designed for high-fidelity amplification of DNA fragments. It contains a DNA polymerase enzyme with proofreading activity, along with necessary buffers and reagents for performing PCR reactions.
Automatically generated - may contain errors
2 protocols using nebnext high fidelity pcr kit
1
ATAC-Seq of AML Cell Lines
2
ATAC-Seq of AML Cell Lines
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Five million cells from MV4;11 and MOLM13 were washed in PBS and resuspend in a hypotonic buffer (50 mM KCl, 10 mM MgSO4, 5 mM HEPES, 0.05% NP-40, 1 mM PMSF, 3 mM DTT) to generate intact nuclei and washed three times in a nuclear wash buffer (10 mM Tris HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2). 50,000 nuclei per cell line were used for the transposition reaction. Nuclei were incubated in 1X TD Buffer (Illumina) and 2.5 μM Transposase enzyme (Illumina) for 30 minutes at 37 °C. Transposed DNA was then extracted from nuclei using the MiniElute PCR purification Kit (Qiagen). Transposed DNA fragments were amplified using NEBNext® High-Fidelity PCR kit (NEB) with a universal Nextera primer and a unique indexing primer and sequenced using NextSeq550 (Illumina) obtaining paired end reads (R1: 75bp; R2: 75bp) with 57 million and 48 million reads obtained for the MOLM13 and MV4;11 cell lines, respectively.
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