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.
Transposase enzyme
Manufactured by Illumina
The Transposase enzyme is a core component of Illumina's library preparation workflows. It is responsible for fragmenting DNA samples and adding adapter sequences, which enables subsequent amplification and sequencing. The Transposase enzyme facilitates this critical step in the library preparation process, enabling the generation of sequencing-ready libraries.
Automatically generated - may contain errors
Lab products found in correlation
Td buffer, by Illumina (3 mentions)
Nextseq 550, by Illumina (2 mentions)
Minielute pcr purification kit, by Qiagen (2 mentions)
Nebnext high fidelity pcr kit, by New England Biolabs (2 mentions)
Hiseq 4000, by Illumina (1 mentions)
Fastqc v0, by Babraham Bioinformatics (1 mentions)
Ampure bead, by Beckman Coulter (1 mentions)
Minelute pcr purification kit, by Roche (1 mentions)
Hifi hotstart readymix pcr kit, by Roche (1 mentions)
3 protocols using transposase enzyme
1
ATAC-Seq of AML Cell Lines
2
ATAC-seq of HUVEC-FUCCI cell cycle
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Subconfluent, HUVEC-FUCCI were lifted and sorted into early G1 and late G1 cell cycle states, then immediately collected for ATAC-sequencing analysis (n = 4 biological replicates). Library preparation was performed as previously described77 (link). Briefly, cells were pelleted by 13,000 rpm for 1.5 min in a tabletop centrifuge, cells were resuspended in 50 μL Transposase Reaction Mix (25 μL 2x TD Buffer, 2.5 μL Transposase Enzyme from Illumina Nextera Cat# FC121-1030, 22 μL nuclease-free water, 0.5 μL 0.1% digitonin) and incubated for 30 min at 37 °C, then DNA was purified using a QIAGEN MinElute PCR Purification Kit (Cat# 28004), amplified using KAPA HiFi HotStart ReadyMix PCR kit, and sequenced tagged primers according to the published protocol, then amplified DNA was purified using AMPure Beads (Beckman Coulter Cat# A63880). Sequencing was performed at the Yale Center for Genomic Analysis (Illumina HiSeq4000). Raw read data was quality controlled with FastQC v0.11.9 (Babraham Bioinformatics), filtered and trimmed with Trimmomatic v0.3378 (link), then peaks were called with MACS2 v2.2.7.179 , and differential peak analysis was performed with HOMER v4.11 (UCSD).
3
ATAC-Seq of AML Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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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