A total of 50,000 cells per replicate were prepared for ATAC-seq libraries using the Omni-ATAC protocol51 (link). To isolate nuclei, cells were resuspended in 50 μl of lysis buffer (0.1% Tween-20, 0.1% NP-40, and 0.01% digitonin in RSB buffer (10 mM Tris–HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2)) and incubated on ice for 3 min. RSB buffer supplemented with 0.1% Tween-20 was used to wash out lysis buffer, and then nuclei were pelleted by centrifugation for 10 min at 500 × g. For transposition, nuclei were resuspended in 50 µl of Transposition mix plus 25 µl of Tagmentation DNA buffer (2.5 µl Tagment DNA enzyme (Illumina), 25 µl of Tagmentation DNA buffer (Illumina), 0.1% Tween-20 and 0.01% Digitonin) and incubated for 30 min at 37 °C. A MinElute PCR purification kit (Qiagen) was used to purify the transposed DNA, and libraries were amplified by PCR with barcoded Nextera primers (Illumina) using 2× NEB Next High-Fidelity PCR Master Mix. The libraries were purified, and sizes selected with AMPure XP beads (Beckman Coulter) for fragments between ~100 and 1000 bp in length according to the manufacturer’s instructions. Paired-end sequencing was performed on an Illumina NextSeq 500 high-output flow cell.
Tagmentation dna buffer
Manufactured by Illumina
Tagmentation DNA buffer is a laboratory reagent used in the process of tagmentation, which is a method for fragmentation and adapter ligation of DNA samples in a single step. The buffer facilitates the efficient fragmentation and adapter attachment required for next-generation sequencing library preparation.
Automatically generated - may contain errors
Lab products found in correlation
Tagment dna enzyme, by Illumina (2 mentions)
Nextseq 500 high output flow cell, by Illumina (2 mentions)
Ampure xp bead, by Beckman Coulter (2 mentions)
Minelute pcr purification kit, by Qiagen (2 mentions)
Tagmentation enzyme, by Illumina (2 mentions)
Bioanalyzer high sensitivity, by Agilent Technologies (2 mentions)
Hifi polymerase, by Roche (2 mentions)
Barcoded nextera primers, by Illumina (1 mentions)
Pure beads, by Roche (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Nextera, by Illumina (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Nextera primers, by Illumina (1 mentions)
5 protocols using tagmentation dna buffer
1
Omni-ATAC-seq Library Preparation Protocol
2
ATAC-seq for Myeloma Cell Analysis
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ATAC-seq was performed on 50,000 viable sorted myeloma cells similar to previously described43 (link),61 (link),62 (link). Briefly, cells were pelleted at 500 × g for 10 min at 4 °C and resuspend in ice-cold nuclei-lysis buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL) and centrifuged at 500 × g for 30 min at 4 °C. Nuclei were resuspended in 22.5 μl of tagmentation DNA buffer (Illumina) with 2 μl tagmentation enzyme (Illumina) at 37 °C for 60 min. Proteins were digested with 2 μg of proteinase K at 40 °C for 60 min. Tagmented DNA was isolated with two rounds of negative (0.6×) and positive (1.2×) size selection with SPRI beads (PureBeads, Kapa Biosystems). ATAC-seq libraries were amplified 12 times with Hifi Polymerase (Kapa Biosystems) and quantitated by qPCR (Kapa Biosystems) and high sensitivity bioanalyzer (Agilent). Sequencing was performed on a NovaSeq 6000 (Illumina) using 150 bp paired-end at the New York Genome Center.
3
Assay for Transposase-Accessible Chromatin
4
ATAC-seq Profiling of Sorted Cells
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ATAC-seq was performed on 3000–5000 sorted cells similarly to that previously described48 (link),66 (link). Briefly, cells were pelleted at 500 × g for 10 min at 4 °C and resuspended in 50 μl nuclei lysis buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL) and centrifuged at 500 × g for 30 min at 4 °C. Nuclei were resuspended in 22.5 μl of tagmentation DNA buffer (Illumina) with 2.5 μl tagmentation enzyme (Illumina) at 37 °C for 60 min. DNA was isolated by proteinase K digestion (2 μg) at 40 °C for 30 min followed by negative (0.6×) and positive (1.2×) size selection with SPRI beads (Agencourt AMPure XP, Beckman Coulter). ATAC-seq libraries were amplified 12–13 times with Hifi Polymerase (Kapa Biosystems) and quantitated by qPCR (Kapa Biosystems) and high sensitivity bioanalyzer (Agilent) and sequenced using 50 bp paired-end sequencing on an Illumina HiSeq 2500 (Illumina) by NYU Genome Technology Center.
5
ATAC-seq protocol for MCTS samples
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ATAC libraries were prepared from separately cultured MCTS samples using the OmniATAC protocol 89 (link) . Briefly, upon MCTS dissociation, cell nuclei were obtained by resuspending cells in 50 µl of lysis buffer (0.1% Tween-20, 0.1% NP-40 and 0.01% Digitonin in RSB buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl 2 )) and incubation on ice for 3 min. The lysis buffer was washed out by RSB buffer supplemented with 0.1% Tween-20. Subsequently, nuclei were pelleted by centrifugation for 10 min at 500g, resuspended in 50 µl of Transposition mix (2.5 µl Tagment DNA enzyme (Illumina), 25 µl of Tagmentation DNA buffer (Illumina), 0.1% Tween-20 and 0.01% Digitonin) and incubated for 30 min at 37 °C. DNA was purified with a MinElute PCR purification kit (Qiagen), and libraries were amplified by PCR with barcoded Nextera primers (Illumina) using 2X NEBNext High-Fidelity PCR Master Mix (NEB). For sequencing, libraries were size-selected with AMPure XP beads (Beckman Coulter) for fragments between ~100 and 1,000 bp in length according to the manufacturer's instructions. Sequencing was performed using paired-end reads on an Illumina NextSeq 500 high output flow cell.
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