ATAC-seq was performed as previously described (Buenrostro et al., 2013 (link)). 60,000 cells were washed once with 100ml PBS and resuspended in 50ml lysis buffer (10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2, 0.2% IGEPAL CA-630). The suspension of nuclei was then centrifuged for 10min at 500g at 4 °C, followed by the addition of 50ml transposition reaction mix (25ml TD buffer, 2.5ml Tn5 Transposase and 22.5ml Nuclease Free H2O) and incubation at 37 °C for 30min. DNA was isolated using MiniElute Kit (QIAGEN). Libraries were amplified by PCR (13 cycles). After the PCR reaction, the library was selected for fragments between 100bp and 1000bp with AmpureXP beads (Beckman Coulter). Libraries were purified with Qiaquick PCR (QIAGEN) and integrity checked on a Bioanalyzer before sequencing.
Qiaquick pcr
Manufactured by Qiagen
Sourced in United States, Germany, Spain
The Qiaquick PCR is a centrifuge-based purification system designed for the rapid and efficient purification of PCR products. It utilizes a silica-membrane technology to selectively bind and purify DNA fragments from complex mixtures, allowing for the removal of primers, nucleotides, and other reaction components. The Qiaquick PCR kit provides a simple and reliable method for the purification of PCR amplicons, enabling downstream applications such as sequencing, cloning, and restriction enzyme digestion.
Automatically generated - may contain errors
Lab products found in correlation
Minelute kit, by Qiagen (8 mentions)
Nextera dna library preparation kit, by Illumina (5 mentions)
Ampure xp bead, by Beckman Coulter (3 mentions)
Nextseq 500 high output kit v2, by Illumina (3 mentions)
Kapa library quantification kit, by Qiagen (3 mentions)
Minielute kit, by Qiagen (2 mentions)
Nextseq 500 high output kit v2 150 cycles, by Illumina (2 mentions)
Novaseq 6000, by Illumina (2 mentions)
Bioanalyzer, by Qiagen (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Gel extraction kit, by Qiagen (1 mentions)
Wizard plus sv minipreps dna purification system, by Promega (1 mentions)
Phusion high fidelity dna polymerase, by New England Biolabs (1 mentions)
Wizard genome dna purification kit, by Promega (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Primestar max dna polymerase, by Takara Bio (1 mentions)
Nucleospin tissue, by Macherey-Nagel (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Gel cleanup kit, by Qiagen (1 mentions)
Trueprep dna library prep kit v2, by Illumina (1 mentions)
18 protocols using qiaquick pcr
1
ATAC-seq protocol for nuclear isolation
2
ATAC-seq Library Preparation Protocol
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ATAC-seq was performed as previously described [7 (link), 19 (link), 20 ]. In brief, a total of 50,000 cells were washed once with 50 μL of cold PBS and resuspended in 50 μL lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.2% (v/v) IGEPAL CA-630). The suspension of nuclei was then centrifuged for 10 min at 500 g at 4 °C, followed by the addition of 50 μL transposition reaction mix (25 μL TD buffer, 2.5 μL Tn5 transposase, and 22.5 μL nuclease-free H2O) of Nextera DNA Library Preparation Kit (96 samples) (FC-121-1031, Illumina). Samples were then PCR amplified and incubated at 37 °C for 30 min. DNA was isolated using a MinElute Kit (QIAGEN). ATAC-seq libraries were first subjected to 5 cycles of pre-amplification. To determine the suitable number of cycles required for the second round of PCR the library was assessed by quantitative PCR as described [20 ], and the library was then PCR amplified for the appropriate number of cycles. Libraries were purified with a Qiaquick PCR (QIAGEN) column. Library concentration was measured by using a KAPA Library Quantification kit (KK4824) according to the manufacturer’s instructions. Library integrity was checked by gel electrophoresis. Finally, the ATAC library was sequenced on a NextSeq 500 using a NextSeq 500 High Output Kit v2 (150 cycles) (FC-404-2002, Illumina) according to the manufacturer’s instruction.
3
ATAC-seq Protocol for Chromatin Profiling
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ATAC-seq was performed as previously described (25 (link)). Briefly, 50,000 cells were washed with 50 μl cold PBS and resuspended in 50 μl lysis buffer (10 mM Tris–HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.2% [vol/vol] IGEPAL CA-630). The suspension of nuclei was then centrifuged for 10 min at 500g at 4°C, followed by the addition of 50 μl transposition reaction mix (25 μl TD buffer, 2.5 μl Tn5 transposase, and 22.5 μl nuclease-free H2O) from the Nextera DNA library Preparation Kit (96 samples) (FC-121-1031; Illumina). Samples were then PCR amplified and incubated at 37°C for 30 min. DNA was isolated using a MinElute Kit (QIAGEN). ATAC-seq libraries were first subjected to five cycles of pre-amplification. To determine the suitable number of cycles required for the next round of PCR, the libraries were assessed by quantitative PCR as described (26 (link)), and the libraries were then PCR amplified for the appropriate number of cycles according to the qRT-PCR results. Libraries were purified with a Qiaquick PCR (QIAGEN) column, and the library’s concentration was measured using a KAPA Library Quantification Kit (KK4824) according to the manufacturer’s instructions. Finally, the ATAC libraries were sequenced on the NextSeq 500 sequencing platform using a NextSeq 500 High Output Kit v2 (150 cycles) (FC-404-2002; Illumina) according to the manufacturer’s instructions.
4
ATAC-seq Protocol for Chromatin Profiling
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ATAC-seq was performed as previously described (25 (link)). Briefly, 50,000 cells were washed with 50 μl cold PBS and resuspended in 50 μl lysis buffer (10 mM Tris–HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.2% [vol/vol] IGEPAL CA-630). The suspension of nuclei was then centrifuged for 10 min at 500g at 4°C, followed by the addition of 50 μl transposition reaction mix (25 μl TD buffer, 2.5 μl Tn5 transposase, and 22.5 μl nuclease-free H2O) from the Nextera DNA library Preparation Kit (96 samples) (FC-121-1031; Illumina). Samples were then PCR amplified and incubated at 37°C for 30 min. DNA was isolated using a MinElute Kit (QIAGEN). ATAC-seq libraries were first subjected to five cycles of pre-amplification. To determine the suitable number of cycles required for the next round of PCR, the libraries were assessed by quantitative PCR as described (26 (link)), and the libraries were then PCR amplified for the appropriate number of cycles according to the qRT-PCR results. Libraries were purified with a Qiaquick PCR (QIAGEN) column, and the library’s concentration was measured using a KAPA Library Quantification Kit (KK4824) according to the manufacturer’s instructions. Finally, the ATAC libraries were sequenced on the NextSeq 500 sequencing platform using a NextSeq 500 High Output Kit v2 (150 cycles) (FC-404-2002; Illumina) according to the manufacturer’s instructions.
5
Construction and Preparation of ssDNA Templates
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Construction of the pHP727FXc plasmid has been described previously (Canceill and Ehrlich, 1996 (link)). Preparation of ssDNA templates was carried out essentially as described (Canceill and Ehrlich, 1996 (link)) with the following modifications: plasmid DNA was extracted using a Maxi Plasmid Kit (Qiagen). Briefly, a specific nick was introduced into the f1 replication origin (+) strand of purified FXc plasmid DNA using the M13 gpII protein. The reaction was stopped with 20 mM EDTA and the products treated with 200 μg/ml proteinase K for 10 min at 55°C, phenol extracted and dialyzed against TE buffer. Nicked strands were removed by exonuclease III digestion (10–40 units per μg of DNA for 1 h at 37°C). Finally, Exo III, nucleotides and oligonucleotides were removed using QIAquick®; PCR (Qiagen) purification kits.
6
Bacterial DNA Isolation and Plasmid Purification
7
Protocol for CRISPR Screening via NGS
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Total DNA was purified from cell pellets by spin column purification (NucleoSpin Tissue, Machery-Nagel), and purified DNAs from all 10–12 selection plates of a given transduction were combined. These pooled DNAs represented all of the cells that survived B[a]P exposure from the 2 × 106–4.8 × 106 cells of a given transduction that were exposed to B[a]P.
Next-generation sequencing (NGS) libraries were prepared by amplifying 317 bp products from the sgRNA regions of integrated GeCKO library viruses using NGS Library Primary PCR primers (Shalem et al., 2014 (link)), 5 µg of pooled template DNA and PrimeSTAR Max DNA polymerase (Takara) in a 50 µl PCR reactions that underwent 18 cycles of amplification. These reaction products were column purified (QIAquick PCR—Qiagen), and barcoded NGS adaptor sequences (Shalem et al., 2014 (link)) were attached by a 24-cycle second round of PCR using 5 µl of purified 1° PCR product as template and single stranded Ultramer DNAs as primers. Standard primers and Ultramer primers were purchased from IDT. NGS reactions were performed on an Illumina HiSeq 2500 platform. Data were exported in qseq format and processed on the UCLA Hoffman2 cluster.
Next-generation sequencing (NGS) libraries were prepared by amplifying 317 bp products from the sgRNA regions of integrated GeCKO library viruses using NGS Library Primary PCR primers (Shalem et al., 2014 (link)), 5 µg of pooled template DNA and PrimeSTAR Max DNA polymerase (Takara) in a 50 µl PCR reactions that underwent 18 cycles of amplification. These reaction products were column purified (QIAquick PCR—Qiagen), and barcoded NGS adaptor sequences (Shalem et al., 2014 (link)) were attached by a 24-cycle second round of PCR using 5 µl of purified 1° PCR product as template and single stranded Ultramer DNAs as primers. Standard primers and Ultramer primers were purchased from IDT. NGS reactions were performed on an Illumina HiSeq 2500 platform. Data were exported in qseq format and processed on the UCLA Hoffman2 cluster.
8
ATAC-seq Protocol for Chromatin Accessibility Analysis
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ATAC-seq was performed as previously described (23 ). In brief, 50,000 cells were collected and washed once with 50 ml cold PBS. Then 50 ml lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.2% (v/v) IGEPAL CA-630) was used to resuspend cells. The suspension was then centrifuged at 500g for 10 min at 4 °C, followed by addition of 50 ml transposition reaction mix (25 ml TD buffer, 2.5 ml Tn5 transposase, and 22.5 ml nuclease-free H2O) of Nextera DNA library Preparation Kit (96 samples) (FC-121-1031, Illumina). After suspension, samples were amplified by PCR and incubated at 37 °C for 30 min. DNA was isolated using a MinElute Kit (QIAGEN). ATAC-seq libraries were subjected to five cycles of preamplification first to determine the number of cycles required for the second round of PCR. Then the amplified libraries, amplified by PCR for an appropriate number of cycles, were purified with a Qiaquick PCR (QIAGEN) column. The concentration of library was measured using a KAPA Library Quantification Kit (KK4824). Library integrity was checked by gel electrophoresis. Finally, the ATAC library was sequenced on a NextSeq 500 using a NextSeq 500 High Output Kit v2 (150 cycles) (FC-404-2002, Illumina).
9
ATAC-seq Protocol for Chromatin Accessibility
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ATAC-seq was performed as previously described [31 (link), 32 ]. Briefly, 50,000 cells were washed with 50 μl cold PBS and resuspended in 50 μl lysis buffer (10 mM Tris–HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.2% (v/v) IGEPAL CA-630). The suspension of nuclei was then centrifuged for 10 min at 500 g at 4 °C, followed by the addition of 50 μl transposition reaction mix (25 μl TD buffer, 2.5 μl Tn5 transposase and 22.5 μl nuclease-free H2O) of Nextera DNA library Preparation Kit (96 samples) (FC-121-1031, Illumina). Samples were then PCR amplified and incubated at 37 °C for 30 min. DNA was isolated using a MinElute Kit (Qiagen). ATAC-seq libraries were first subjected to 5 cycles of pre-amplification. To determine the suitable number of cycles required for the next round of PCR, the libraries were assessed by quantitative PCR as described [32 ], and the libraries were then PCR amplified for the appropriate number of cycles according to the qPCR results. Libraries were purified with a Qiaquick PCR (Qiagen) column, and the libraries concentration were measured using a KAPA Library Quantification kit (KK4824) according to the manufacturer’s instructions. Finally, the ATAC libraries were sequenced on NextSeq 500 sequencing platform using a NextSeq 500 High Output Kit v2 (150 cycles) (FC-404-2002, Illumina) according to the manufacturer’s instructions.
10
ATAC-seq Protocol for Chromatin Profiling
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ATAC-seq was performed as previously described (Buenrostro et al. 2013 (link)). Sixty-thousand cells were washed once with 100 mL of PBS and resuspended in 50 mL of lysis buffer (10 mM Tris-HCl at pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.2% IGEPAL CA-630). The suspension of nuclei was then centrifuged at 500g for 10 min at 4°C, followed by the addition of 50 mL of transposition reaction mix (25 mL of TD buffer, 2.5 mL of Tn5 transposase, 22.5 mL of nuclease-free H2O) and incubation for 30 min at 37°C. DNA was isolated using MiniElute kit (Qiagen). Libraries were amplified by PCR (13 cycles). After the PCR reaction, the library was selected for fragments between 100 and 1000 bp with AMPure XP beads (Beckman Coulter). Libraries were purified with Qiaquick PCR (Qiagen) and integrity-checked on a Bioanalyzer before sequencing.
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