50,000 cells per sample underwent tagmentation reaction with Nextera Tn5 transposase using the Illumina Nextera kit and purified with a Qiagen MinElute reaction kit48 (link). The DNA was then PCR amplified for 9 cycles to add indexing primers. SPRI AMPure beads enriched for fragments under ~600 bps. The DNA was cycled again with the 9-cycle protocol, followed by cleanup with SPRI AMPure beads. The DNA was quantified with a Qubit DNA High Sensitivity assay and analyzed for quality and size distribution on an Agilent TapeStation with a High Sensitivity D5000 ScreenTape. Samples were pooled for a 10 nM final overall concentration. Sequencing was performed with an Illumina HiSeq 2500 system with 2 × 50 bp read length by the Washington University in St. Louis School of Medicine Genome Technology Access Center. ATAC-Seq data are available on GEO under GSE145551.
Minelute reaction kit
Manufactured by Qiagen
The MinElute reaction kit is a laboratory tool designed for the purification and concentration of DNA fragments. It utilizes a silica-based membrane technology to efficiently capture and purify DNA samples from various reaction mixtures, enabling users to concentrate their samples for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2500 system, by Illumina (3 mentions)
High sensitivity dna assay, by Agilent Technologies (3 mentions)
D5000 screentape, by Agilent Technologies (3 mentions)
Nextera kit, by Qiagen (3 mentions)
Tapestation, by Agilent Technologies (2 mentions)
Quick gdna miniprep kit, by Zymo Research (2 mentions)
Pan monocyte isolation kit, by Miltenyi Biotec (2 mentions)
Tapestation 2200, by Agilent Technologies (1 mentions)
5 protocols using minelute reaction kit
1
Transposase-based ATAC-Seq library preparation
2
Monocyte Chromatin Accessibility and Methylation
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Cryopreserved PBMCs were thawed, washed, and resuspended at 2E6 cells/mL in RPMI-10 supplemented with M-CSF (50 ng/mL), then rested overnight in 6-well non-TC treated dishes. CD14+/CD16+ monocytes were isolated by negative selection magnetic bead column purification (Pan Monocyte Isolation Kit, Miltenyi Biotec). For chromatin accessibility (ATAC-seq), 5×10E4 monocytes were removed and lysed in resuspension buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2) plus 0.1% NP40, 0.1% Tween-20, and 0.01% digitonin. Cells were lysed on ice for 3 minutes and then washed in resuspension buffer plus 0.1% Tween-20. Nuclei were centrifuged for 10 min at 500×g and 4°C. Supernatant was removed and nuclei were resuspended in 50 μL of transposition mix (Tagment DNA Buffer, 0.1% Tween-20, 0.05% digitonin, 2.5 μL Tagment DNA Enzyme). Transposition reactions were incubated for 30 minutes at 37°C with shaking at 10E3 RPM. DNA fragments were purified with MinElute Reaction kit according to the manufacturer’s instructions (Qiagen). For methylation, monocytes were plated at 5×10E5 per well in RPMI-10 supplemented with M-CSF. After 24 hours, media was removed, and genomic DNA was isolated using Quick-gDNA MiniPrep kit according to the manufacturer’s instructions (Zymo Research).
3
Monocyte Chromatin Accessibility and Methylation
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Cryopreserved PBMCs were thawed, washed, and resuspended at 2E6 cells/mL in RPMI-10 supplemented with M-CSF (50 ng/mL), then rested overnight in 6-well non-TC treated dishes. CD14+/CD16+ monocytes were isolated by negative selection magnetic bead column purification (Pan Monocyte Isolation Kit, Miltenyi Biotec). For chromatin accessibility (ATAC-seq), 5×10E4 monocytes were removed and lysed in resuspension buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2) plus 0.1% NP40, 0.1% Tween-20, and 0.01% digitonin. Cells were lysed on ice for 3 minutes and then washed in resuspension buffer plus 0.1% Tween-20. Nuclei were centrifuged for 10 min at 500×g and 4°C. Supernatant was removed and nuclei were resuspended in 50 μL of transposition mix (Tagment DNA Buffer, 0.1% Tween-20, 0.05% digitonin, 2.5 μL Tagment DNA Enzyme). Transposition reactions were incubated for 30 minutes at 37°C with shaking at 10E3 RPM. DNA fragments were purified with MinElute Reaction kit according to the manufacturer’s instructions (Qiagen). For methylation, monocytes were plated at 5×10E5 per well in RPMI-10 supplemented with M-CSF. After 24 hours, media was removed, and genomic DNA was isolated using Quick-gDNA MiniPrep kit according to the manufacturer’s instructions (Zymo Research).
4
Transposase-based DNA Library Preparation
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Up to 50,000 cells per sample were tagmented with Nextera Tn5 transposase using the Illumina Nextera kit and purified with a Qiagen MinElute reaction kit per methods modified from Buenrostro et al. (2013) (link). The DNA was then PCR amplified to add indexing primers in nine cycles. SPRI AMPure beads enriched for fragments under ∼600 bp. The library was then amplified again with the nine-cycle protocol, followed by cleanup with SPRI AMPure beads. The DNA library was quantified with a Qubit DNA High Sensitivity assay and analyzed for quality and size distribution on an Agilent 2200 TapeStation with a High Sensitivity D5000 ScreenTape. Samples were pooled at a 10 nM final concentration. Sequencing was run with an Illumina HiSeq 2500 system with 2 × 50 bp read length by the Washington University in St Louis School of Medicine GTAC.
5
Zebrafish Bulk Melanoma Tumor ATAC-Seq
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Following humane euthanasia, grossly visible bulk melanoma tumors from Tg(BRAF V600E / crestin:EGFP);p53 -/-zebrafish were excised with a razor, manually sheared, and incubated in fresh 0.9X PBS with 2.5 mg/mL Liberase for up to 30 minutes to dissociate cells. Fetal bovine serum addition terminated the digestion, and the cells were passed through a 40 mm filter. Cells were centrifuged at 2000 x g for 5 minutes at 4 o C. Supernatant was removed and the cells were resuspended in 500 µL of 0.9X PBS and kept on ice. ATAC-Seq 50,000 cells per sample underwent tagmentation reaction with Nextera Tn5 transposase using the Illumina Nextera kit and purified with a Qiagen MinElute reaction kit (Buenrostro et al., 2015) .
The DNA was then PCR amplified for 9 cycles to add indexing primers. SPRI AMPure beads enriched for fragments under ~600 bp. The DNA was cycled again with the 9-cycle protocol, followed by cleanup with SPRI AMPure beads. The DNA was quantified with a Qubit DNA High Sensitivity assay and analyzed for quality and size distribution on an Agilent TapeStation with a High Sensitivity D5000 ScreenTape. Samples were pooled for a 10 nM final overall concentration. Sequencing was performed with an Illumina HiSeq 2500 system with 2 x 50 bp read length by the Washington University in St. Louis School of Medicine Genome Technology Access Center.
The DNA was then PCR amplified for 9 cycles to add indexing primers. SPRI AMPure beads enriched for fragments under ~600 bp. The DNA was cycled again with the 9-cycle protocol, followed by cleanup with SPRI AMPure beads. The DNA was quantified with a Qubit DNA High Sensitivity assay and analyzed for quality and size distribution on an Agilent TapeStation with a High Sensitivity D5000 ScreenTape. Samples were pooled for a 10 nM final overall concentration. Sequencing was performed with an Illumina HiSeq 2500 system with 2 x 50 bp read length by the Washington University in St. Louis School of Medicine Genome Technology Access Center.
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