ATAC-seq was performed as previously described (Buenrostro et al., 2013 (link)) with minor adaptations. Oli-neu cells (differentiated, siRNA treated as described above) were collected with Accutase solution (Sigma, A6964). 50 000 cells per condition were lysed in lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1% IGEPAL CA-630) and centrifuged at 500xg at 4°C for 20 minutes. The cells were then resuspended in tagmentation mix (2.5 μL TD buffer, 2.5 μL Tn5 enzyme, Illumina) and the DNA was transposed for 30 minutes at 37°C, where after the DNA was purified using the QIAGEN MinElute kit. After PCR amplification with 7-8 cycles (cycles determined with qPCR) the libraries were sequenced on a HiSeq2500 Illumina sequencer. Three replicates per condition were performed in different days and different passages. Each replicate had >10 million reads with an average of 45 million reads per sample.
Tn5 enzyme
Manufactured by Illumina
The Tn5 enzyme is a transposase protein derived from the Tn5 transposon. It functions by binding to DNA and catalyzing the insertion of DNA fragments into target DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (6 mentions)
Minelute kit, by Qiagen (4 mentions)
Td buffer, by Illumina (4 mentions)
Glycine, by Thermo Fisher Scientific (4 mentions)
Dynabeads myone streptavidin c1, by Thermo Fisher Scientific (4 mentions)
Biotin 14 datp, by Jena Biosciences (4 mentions)
Nextseq 500, by Illumina (4 mentions)
Minelute pcr purification kit, by Qiagen (3 mentions)
Mboi restriction enzyme, by New England Biolabs (3 mentions)
Dpnii restriction enzyme, by New England Biolabs (3 mentions)
Nextera xt library sample preparation kit, by Illumina (3 mentions)
Nextera tagmentation dna buffer, by Illumina (3 mentions)
Neutralize tagment buffer, by Illumina (3 mentions)
Nextera npm mix, by Illumina (3 mentions)
Formaldehyde, by Thermo Fisher Scientific (3 mentions)
Formaldehyde, by Merck Group (2 mentions)
Novaseq 6000 platform, by Illumina (2 mentions)
Phenol chloroform, by Merck Group (2 mentions)
Tagment dna buffer, by Illumina (2 mentions)
Ampure xp bead, by Beckman Coulter (2 mentions)
20 protocols using tn5 enzyme
1
Profiling Chromatin Accessibility in Oligodendrocytes
2
Transposase-based Chromatin Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Embryo halves were then homogenized using Kimble Kontes Pellet Pestle (cat no. K749521-1590). IGEPal CA-630 was added to a final concentration of 0.1%. After a 10 minute incubation, nuclei were spun down and resuspended in water. Twenty halves were added to the transposition reaction containing 25ul of 2x TD buffer (Illumina), and 7.5ul of Tn5 enzyme (Illumina). The reaction was incubated at 37°C for 30 minutes. Transposed DNA was purified using Qiagen Minelute kit. Libraries were then amplified using Phusion (NEB cat no. F531S) and Illumina Nextera index kit (cat no. FC-121-1011). Libraries were then purified with Ampure Beads at a 1.2: 1 beads to sample ratio and sequenced on the Hiseq4000 using 100bp paired end reads. Fragments over 500bp were removed from libraries using a Pippen prep to reduce sequencing bias with the Hiseq4000.
3
Low-Cost Single-Cell ATAC-Seq Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For Pi-ATAC, reagent costs is largely driven by the PCR master mix. Cost estimate per cell: Tn5 enzyme (Illumina, $0.00049 per cell, assuming 2.5 µl Tn5 per 50,000 cells), NEBNext® High-Fidelity 2X PCR Master Mix (NEB, M0541) and primers (~$1.30 per cell) and negligible costs for antibodies per cell (about $3 per protein per 106 cells). In summary, it costs about $1.50 per cell with 50 µl PCR reaction system. In addition, we had succeeded with low-scale volume PCR reaction system at 5 µl per well of a 96-well plate, where the cost is ~$0.15 per cell.
4
HiChIP Profiling of H3K27Ac and RelA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HiChIP was performed as previously described53 (link), using antibodies against 1 μg of H3K27ac (Abcam) or 5 μg of RelA (Cell Signaling Technology). Briefly, cells (2 × 106 for H3K27ac, 1 × 107 for RelA) were crosslinked with 1% formaldehyde (Sigma) for 10 min and subsequently quenched with 0.125 M glycine (Invitrogen). Chromatin was digested using MboI restriction enzyme (NEB), followed by biotin incorporation with Biotin-14-dATP (Jena bioscience) in end-repair step, ligation, and sonication. Sheared chromatin was then incubated with antibodies recognizing H3K27ac or RelA at 4 °C overnight. Chromatin-antibody complexes were captured by Protein A and G magnetic bead (Invitrogen) and subsequently washed with Low Salt Wash Buffer, High Salt Wash Buffer, and LiCl Wash Buffers before being eluted. DNA was purified with the MinElute PCR Purification Kit (Qiagen) and quantified using the Qubit dsDNA HS Assay Kit (Invitrogen). Subsequently, 50–150 ng was used for capture with Dynabeads MyOne Streptavidin C-1 (Invitrogen) and an appropriate amount of Tn5 enzyme (Illumina) was added to captured DNA to generate sequencing library. Each library was paired-end sequenced (100 bp) on Illumina NovaSeq6000 platform. Two biological replicates were performed for each condition.
5
In situ Hi-C for Chromatin Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In situ Hi-C was performed as previously described.72 (link) In brief, liver samples were harvested, flash-frozen in liquid nitrogen, and pulverized before 1% formaldehyde cross-linking for 20 minutes and subsequent quenching with 0.125 mol/L glycine. Liver nuclei were isolated using a sucrose cushion after cross-linked tissues were thawed and dissociated using a gentleMACS Tissue Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). Chromatin was digested using DpnII restriction enzyme (New England Biolabs, Ipswich, MA), followed by biotin incorporation with Biotin-14-Deoxyadenosine triphosphate (dATP) (Jena bioscience, Löbstedter Str. 71, 07749 Jena, Germany). After de–cross-linking, ligated DNA was purified and sheared to 200 to 300 bp. DNA was purified with phenol/chloroform (Sigma-Aldrich, St.Louis, MO) and quantified using Qubit dsDNA HS Assay Kits (Thermo Fisher Scientific, Waltham, MA). A total of 150 ng was used for capture with Dynabeads MyOne Streptavidin C-1 (Thermo Fisher Scientific, Waltham, MA), and an appropriate amount of Tn5 enzyme (Illumina, San Diego, CA) was added to captured DNA to generate sequencing libraries. Each library was paired-end sequenced (101 bp) on an Illumina NovaSeq6000 platform. Two biological replicates were performed in each condition.
6
ATAC-Seq Protocol for Early Embryos
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Early nc14 embryos were placed in ATAC-seq lysis buffer (Buenrostro et al., 2013 (link)) without detergent, with 5% glycerol added. Embryos were then taken out of the freezing solution and placed onto a glass slide which was then put on dry ice for 2 min. Once embryos were completely frozen, the glass slide was removed and embryos were sliced with a razor blade chilled in dry ice. Once sliced embryo halves were moved to tubes containing ATAC-seq lysis buffer with 0.15 mM spermine added to help stabilize chromatin. Embryo halves were then homogenized using single use plastic pestles. IGEPal CA-630 was added to a final concentration of 0.1%. After a 10 min incubation nuclei were spun down and resuspended in water. Twenty halves were added to the transposition reaction containing 25 µl of 2x TD buffer (Illumina), and 2.5 ul of Tn5 enzyme (Illumina) and the reaction was incubated at 37°C for 30 min as in Buenrostro et al. (2013) (link). Transposed DNA was purified using Qiagen Minelute kit. Libraries were then amplified using phusion 2x master mix (NEB) and indexed primers from Illumina. Libraries were then purified with Ampure Beads and sequenced on the Hiseq4000 using 100 bp paired end reads.
7
Bulk ATAC-seq Library Preparation
8
ATAC-seq protocol for chromatin accessibility
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ATAC-seq was performed following a protocol from the Kaestner Lab (https://www.med.upenn.edu/kaestnerlab/assets/user-content/documents/ATAC-seq%20Protocol%20(Omni)%20-%20Kaestner%20Lab.pdf ) with minor modifications. A total of 100,000 Kc cells were washed with 50 μL cold 1X PBS. Cell pellet was lysed with 50 μL cold lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40, 0.1% Tween-20, 0.01% Digitonin) and incubated for 10 min on ice. Then 500 μL of wash buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20) was added to lysate. Nuclei were then collected by centrifuging at 500 × g for 10 min at 4 °C and nuclei were resuspended in 50 μL of transposition reaction mix [25 μL 2X TD buffer (Illumina), 16.5 μL PBS, 0.5 μL 10% Tween-20, 0.5 μL, 0.5 μL 1% Digitonin, 2.5 uL Tn5 enzyme (Illumina), 5 μL nuclease-free water] and incubated for 45 min at 37 °C at 1000 rpm (Eppendorf Thermomixer) for fragmentation. DNA was purified with Qiagen Minelute columns, and libraries were amplified by adding 10 μL DNA to 25 μL of NEBNext HiFi 2x PCR mix (New England Biolabs) and 2.5 μL of 25 μM each of Ad1 and Ad2 primers using 11 PCR cycles. Libraries were purified with 1.2× AMPure XP beads. All samples were sequenced with NextSeq-550 (Illumina) using 50 bp paired-end sequencing.
9
ATAC-seq protocol for chromatin accessibility
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ATAC-seq was performed similar to that previously described (21, 58 (link)). Briefly, 50,000 viable cells were isolated by FACS. Nuclei were isolated by spinning at 500 g for 10 minutes, all supernatant was pipet decanted, and pelleted cells were resuspended in 25 μL Tagmentation Reaction Mix composed of 12.5 μL Tagment DNA Buffer (Illumina), 0.02% Digitonin, 0.1% Tween-20, 2.5 μL TN5 enzyme (Illumina). Tagmentation was performed for 1 hour at 37°C. Tagmented DNA was isolated by adding Tagmentation Clean-up Buffer (326 mmol/L NaCl, 109 mmol/L EDTA, 0.63% SDS, 20 μg Proteinase K) and incubating at 40°C for 1 hour and both a negative (0.7×) and positive (1.2×) SPRI bead (Kapa) selection. Libraries were amplified 12 times with Hifi polymerase (Kapa) and Nextera barcoded primers (Illumina) prior to 1× SPRI bead clean-up and sequencing on a Novaseq (Illumina).
10
Tn5-Based OMNI-ATAC Library Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tn5 transposition was performed using the OMNI-ATAC protocol56 (link). A 2.5 μl volume of Tn5 enzyme (Illumina 20034197) was used in the transposition reaction. Libraries were prepared with NEBNext High-Fidelity 2× PCR Master Mix (NEB M0541L), following the standard protocol. After the initial five cycles of amplification, another four cycles were added, on the basis of qPCR optimization. Following amplification, libraries were size selected (0.5×–1.8×) twice with AMPure XP beads (Beckman Coulter A63880) to remove residual primers and large genomic DNA. Individually barcoded libraries were multiplexed and sequenced with paired-end 76-bp reads on an Illumina NextSeq, using either the Mid or High Output Kit.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!